Transgenic animal model of mood disorders

ABSTRACT

A non-human transgenic animal having a polynucleotide encoding a PTN polypeptide, which polynucleotide is operably linked to a promoter, wherein said transgenic animal has greater than wild-type expression of the PTN polypeptide in at least one brain region, as well as related vectors, methods of producing transgenic animals, in vitro and in vivo screening methods for potential therapeutic agents, and methods for treating and diagnosing neuropsychiatric illnesses, particularly anxiety and depression, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 14/386,319,filed Sep. 18, 2014, which is the U.S. National Stage of InternationalApplication No. PCT/EP2013/055253, filed Mar. 14, 2013, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of GB Application No. 1204816.1, filed Mar. 19, 2012, all ofwhich are incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a marker for psychiatric illness,particularly mood disorders such as Major Depressive Disorder (MDD),Anxiety and Bipolar Disorder (BD), to animal models utilising themarker, to methods of screening for agents that affect the marker andwhich may have therapeutic potential, and to uses for treatment based ondown-regulation of the marker expression and/or antagonism of itsactivity.

BACKGROUND TO THE INVENTION

Psychiatric disorders such as major depression, anxiety or bipolardisorder are becoming one of the major public-health problems on aglobal scale. The causes of these disorders are as yet poorlyunderstood, although genetic factors undoubtedly play an important rolein their aetiology. Furthermore, these illnesses do not develop becauseof the alteration of one single gene but more likely due to changes in agroup of genes, which makes them multi-factor illnesses.

Mood disorders such as major depression, anxiety and bipolar disordersare the most common psychiatric disorders in modern society. About 16%and 1% of the population are estimated to be affected by majordepression and bipolar disorder one or more times during their lifetime,respectively. Such disorders affect approximately 121 million peopleworldwide, with 20% of women and 12% of men estimated to experience adepressive episode in any 1-year period and with evidence of suicidalityin 15% of those affected. In this context, major depression has beenprojected to become the second leading cause of disability worldwide by2020 (second to ischemic heart disease) (Lee et al., 2010)(www.who.int).

Depression is related to the normal emotions of sadness and bereavement,but it does not remit when the external cause of these emotionsdissipates, and it is disproportionate to their cause. It is defined byepisodes of depressed mood lasting for greater than 2 weeks accompaniedby additional symptoms including disturbed sleep and appetite, reducedconcentration and energy, excessive guilt, slowed movements and suicidalthoughts. Also, depression is associated with considerable morbidity andexcess mortality (Sullivan et al., 2000; Belmaker and Agam, 2008).Interestingly, approximately one-third of patients suffering from MDDare refractory to any kind of antidepressant treatment.

The resulting cost to the US economy is estimated to be in the range ofUS $100 billion per year. But in spite of these considerable financialincentives and the public health importance of treating depression,there is an absence of conceptually novel antidepressants on the market,reflecting the difficulties encountered in the development of newtreatment strategies for this common and complex disorder.

Despite extensive investigations, the exact mechanisms responsible forMDD have not been identified. In this context, several hypotheses havebeen proposed to explain the aetiology of MDD. The monoamine hypothesesof depression, which posits that depression is caused by decreasedmonoamine function in the brain, originated from early clinicalobservations. The serendipitous discovery of antidepressants in the1950s has profoundly inspired this hypothesis of the pathogenesis ofdepression. The well-known pharmacological effects of antidepressants onpresynaptic uptake transporters and degradating enzymes (i.e., MAO) ofserotonin and norepinephrine has focused research on causality andtreatment of depression on the metabolism of functional biogenic aminesand the capacity of their respective receptors to alter intracellularsignaling pathways that ultimately induce changes in gene activity.Although these mono amine-based agents are potent antidepressants, andalterations in central monoamine function might contribute marginally togenetic vulnerability, the cause of depression is far from being asimple deficiency of central monoamines. Monoamine oxidase inhibitorsand SSRIs produce immediate increases in monoamine transmission, whereastheir mood-enhancing properties require weeks of treatment. Conversely,experimental depletion of monoamines can produce a mild reduction inmood in unmedicated depressed patients, but such manipulations do notalter mood in healthy controls. It is now thought that acute increasesin the amount of synaptic monoamines induced by antidepressants producesecondary neuroplastic changes that are on a longer timescale andinvolve transcriptional and translational changes that mediate molecularand cellular plasticity (Krishnan and Nestler, 2008)

Alternative hypotheses for MDD have recently provided the rationale forthe emergence of new strategies for antidepressant drug development.These new strategies could indicate that researchers and pharmaceuticalcompanies are now considering the various alternative hypotheses for MDDas complementary rather than competing. Such a position could bring abroader recognition of the heterogeneity of MDD, thereby fostering anexpansion of treatment approaches (Wong and Licinio, 2004).

In recent years, it has been postulated that depression and anxiety maybe caused by cytokine secretion associated with activation of the immunesystem. This hypothesis was proposed initially because the incidence ofimmune abnormalities is higher in depressed and anxious patients than inthe general population, and because depression is a common side effectof cytokine therapy. Cytokines, which are humoral mediators of innateand adaptive immunity, are also important modulators of mood. Cytokinereceptors within the central nervous system are activated by bothperipherally and centrally synthesized cytokines. Low doses oflipopolysaccharide or interleukin 1 (IL-1) produce ‘sickness behaviour’in rodents (consisting of social withdrawal and decreased exploratoryand sexual behaviour), brought about by the release of pro-inflammatorycytokines such as interferon-α, tumour necrosis factor-α(TNF-α), IL-6and IL-1β, which activate the hypothalamic-pituitary-adrenal axis andcentral monoamine systems. There are many similarities between sicknessbehavior and the symptoms of depression. Thus it was suggested thatcytokines could induce depression, or indeed were the cause ofdepression. This hypothesis has come to be called the cytokinehypothesis of depression. Studies found that inflammatory cytokineslevels decreased with various antidepressants treatments, suggestingthan antidepressants may reduce inflammation. Conversely,anti-inflammatory drugs can have an antidepressant effect. Such datasupport the notion that inflammation may in some way contribute to thegenesis of depressive symptoms (Dunn et al., 2005; DellaGioia andHannestad, 2010).

Animal models are widely used to study the neurobiological mechanisms ofdepression and anxiety but mouse models that mimic the full phenotypicspectrum of a psychiatric disorder, such as major depression, arevirtually impossible. However, a recreation of some phenotypiccomponents is feasible and animal models of this disease often try tomimic some symptoms of the disorder. Ideal animal depression models mustbe reasonably analogous to the human symptoms, be able to be monitoredobjectively, be reversed by the same treatment modalities as humans, andbe reproducible between laboratories. Interestingly, antidepressanttreatment has been shown to affect the behavioural responses in thesemodels indicating that certain depression paradigms arepharmacologically sensitive, and therefore, can be used in the testingof antidepressant drugs in mice. In this context, animal models have acentral role in discovering the causes of psychiatric disorders andgenerating novel mechanism-based treatments. One of the most importantadvances in understanding psychiatric disorders has been the developmentof mice with genetically altered expression of a specific protein. Thesenew tools have the potential to examine novel targets for antidepressantactivity. Additionally, these mice will enable better testing of thevalidity of current molecular theories of depression. However, it isdifficult to prepare a model reflecting a human disease having amechanism of development which is unknown, such as depression (Krishnanand Nestler, 2010).

There is at present an unmet need for animal models of psychiatricdiseases such as major depression and/or anxiety. A significantdifficulty remains the translation of genetic association studies intomodels that are effective for evaluating the efficacy of candidatetherapeutic compounds. In particular, the complexity of the disordermeans that it is not normally possible to predict whether a geneticalteration associated with the disorder has a causal role and willproduce behavioural changes of relevance to depression and/or anxiety.

DISCLOSURE OF THE INVENTION

The present inventors have now found that Pleiotrophin (PTN or HeparinBinding Growth Associated Molecule: HB-GAM) expression is altered in theprefrontal cortex of depressed patients who died by suicide, whencompared with samples taken from the same brain region of controlindividuals with no history of mental illness and who died accidentally.Pleiotrophin is a 136 amino acid secreted heparin-binding cytokine(Milner et al., 1992). Ptn gene is expressed in cells in earlydifferentiation stages during several developmental periods. Also, Ptnis over-expressed in different types of cancer (Kadomatsu and Muramatsu,2004). PTN responses are mediated by different receptors: PTNinactivates the receptor protein tyrosine phosphatase (RPTP) 13/cinducing its dimerization. PTN stimulates phosphorylation (activation)of Anaplastic Lymphoma Kinase (ALK) (a tyrosine receptor kinase) throughthe PTN/RPTPIβ/ζ signaling pathway. PTN can also interact with theSyndecan-3 receptor. The interaction of PTN with RPTPβ/ζ, disruptsnormal association of β-catenin with E-cadherin (Deuel et al., 2002;Furuta et al., 2004).

Furthermore, the findings disclosed herein indicate that enhancedexpression of PTN protein is not merely a consequential changeassociated with depression and/or anxiety, but appears to have a causalrole, as shown by depressive-related behavioural changes in transgenicmice that overexpress PTN in brain. The difference observed in PTNlevels in drug-treated and drug-free depressive patients makes PTN anattractive drug target and screening tool for candidate therapeutics forpsychiatric conditions, including depression and/or anxiety.Interestingly, some of the principal players involved in PTN mediatedsignalling have been found altered in Major Depression.

Accordingly, in a first aspect the present invention provides anon-human transgenic animal having a polynucleotide encoding a PTNpolypeptide, which polynucleotide is operably linked to a promoter,wherein said transgenic animal has greater than wild-type expression ofthe PTN polypeptide in at least one brain region. The transgenic animalexhibits one or more anxious and/or depressive-related behaviours, forexample: reduced time spent in open arms of an elevated plus maze;reduced time in central area in an open-field test; reduced time inlight in a light-dark box test; increased latency to feed in a NoveltySuppressed Feeding Test, increased immobility time in tail suspensiontest; and reduced sucrose intake in sucrose intake task.

The transgenic mice described in detail herein were created withinclusion of a neuronal specific promoter, the Thy promoter, which ismore neuronal specific than PDGF promoter which is expressed also inother tissues (Tesseur et al., 2000). The PDGF promoter was used tocreate transgenic mice that also over express PTN not only in neurons(Pavlov et al., 2002). Moreover, the PTN mice of Pavlov et al., 2002,were found to exhibit decreased anxiety in the elevated plus maze test(i.e. more entries on the open arms). This is in marked contrast to thepresent findings, wherein the PTN mice of the present invention, asdescribed in detail herein, were found to exhibit greater anxiety-likebehaviour than wild-type controls (e.g. spending less time, and walkingless distance, in the open arm). The choice of promoter and thecorresponding tissue expression of the PTN transgene has an importanteffect on the behaviour of the PTN transgenic animal, such that the PTNmice of Pavlov et al., 2002, are unsuited to use as a model forpsychiatric illness, particularly disorders of mood, whereas the PTNtransgenic animal of the present invention, having moreneuronal-specific expression of the PTN transgene as compared with thePTN mice of Pavlov et al. 2002, preferably exhibit at least one anxietyand/or depressive-like behaviours, e.g. one or more anxiety and/ordepression-related behaviours selected from: reduced motor activity inan open-field test; reduced time spent in open arms of an elevated plusmaze; reduced time spend in the lit portion of a light-dark box;increased latency to feed in a Novelty Suppressed Feeding Test;increased immobility time in tail suspension test; and decreased sucroseintake. Without wishing to be bound by any particular theory, thepresent inventors believe that the greater neuronal-specificity of theThy1 promoter employed herein as compared with the PDGF beta promoteremployed by Pavlov et al., 2002, contributes to the anxiety and/ordepression-related behaviour of the transgenic animal of the presentinvention.

The PTN transgenic mice described in the Examples herein, having the PTNgene under the control of a Thy1 promoter, exhibit up-regulation of PTNexpression in multiple brain regions that is in the range approximately2.1 to as high as 6.4-fold higher than wild-type littermates (PTN ratioof TG (transgenic) to WT (wild-type). This is a greater level ofover-expression than that reported for Pavlov et al., 2002(approximately 2-fold overexpression in the hippocampus as determined byWestern Blot). Greater neuronal expression of another gene, humanApolipoprotein E4, has been reported when the Thy1 promoter is comparedwith the PDGF beta promoter (Tesseur, et al., 2000).

Further examples of Neuron-Specific Promoters that can be used to createtransgenic mice, in accordance with the present invention, thatoverexpress PTN specifically in brain include (see Table 4): Neuronspecific enolase (NSE); Rhombotin I; PGK; Neurofilament Low (NF-L);dopamine beta-hydroxylase (DBH); Synapsin-1.

The expression of PTN in mouse brain under mouse Thy1 promoter induces adepressive-anxious-like phenotype. In contrast previously described byPavlov et al., when human PDGF promoter is used to drive PTNoverexpression in mice, this phenotype is not replicated. Interestingly,animals with overexpression of a target gene under Thy1 promoter, showedhigher expression levels than those with human PDGF promoter (Tesseur etal., 2000). Furthermore, the Thy1 promoter shows a brain tissue-specificexpression pattern, focusing mainly in cortex and the hippocampus, twokey regions in the development of Mood Disorders, whereas the PDGFpromoter shows a generalized expression in the brain and even in otherorgans (Tesseur et al., 2000). Without wishing to be bound by anyparticular theory, it is presently believed that these two differencesmay be key because the more specific and higher expression of the targetgene, the greater the possibility to obtain a model that mimics moresevere symptoms of the disease as well as more specificity in obtainedphenotypes. Finally, the use of mouse promoter may also be key to obtaina better animal model of Mood Disorders comparing with the PDGF promoterof human origin, due to the advantage of using a DNA sequence with thesame origin as the expression machinery to be used.

The transgenic animal of the invention may contain a foreign gene orpromoter (i.e. genetic material from another species) or it may notcontain any foreign gene or promoter. The latter case is consideredtransgenic herein by virtue of an alteration in the location, copynumber or sequence of a PTN-encoding polynucleotide and/or analternation in the promoter controlling expression of the PTN-encodingpolynucleotide. In certain cases, the transgenic animal of the inventionmay have said polynucleotide encoding a PTN polypeptide present in ahigher than wild-type copy number. For example, the transgenic animalmay carry a native or non-native ptn gene in higher than diploid copynumber. The transgenic animal of the invention may have thepolynucleotide encoding a PTN polypeptide operably linked to a promoterwhich is other than a ptn gene promoter and which is other than thehuman PDGF beta-chain promoter. Thus, for example, the promoter may bean endogenous or exogenous promoter from another gene, preferably apromoter from a gene which exhibits brain-specific or largelybrain-specific expression. In accordance with this and other aspects ofthe present invention, the promoter may be selected from the groupconsisting of: the human or mouse Thy1 gene promoter; human or mouseNeuron specific enolase (NSE); human or mouse Rhombotin I; PGK; human ormouse Neurofilament Low (NF-L); human or mouse dopamine beta-hydroxylase(DBH); human or mouse Synapsin-1.

In certain cases of the transgenic animal according to the inventionsaid polynucleotide encodes a PTN polypeptide which is a mouse, rat orhuman PTN polypeptide, a variant (such as a splice variant), precursor(such as the 168 amino acid human pleiotrophin precursor sequenceavailable at NCBI accession number NP_002816; GI: 4506281 or the 168amino acid mouse pleiotrophin precursor sequence available at NCBIaccession number NP_032999; GI: 6679543), derivative (such as apost-translationally processed polypeptide), homologue or orthologuefrom another species (preferably a mammalian homologue or orthologue) orfragment. The PTN polypeptide or fragment thereof preferably exhibitsbiological activity, particularly the ability to bind to its specificreceptors such as RPTPβ/ζ, ALK or Syndecan 3. In preferred cases of thetransgenic animal according to the invention said polynucleotideencodes:

-   -   (i) a PTN polypeptide having an amino acid sequence having at        least 80%, 90%, 95% or 99% amino acid sequence identity to the        sequence of SEQ ID NO: 2;    -   (ii) a PTN polypeptide having the amino acid sequence of SEQ ID        NO: 2    -   (iii) a PTN polypeptide having an amino acid sequence having at        least 80%, 90%, 95% or 99% amino acid sequence identity to the        sequence of SEQ ID NO: 4;    -   (iv) a PTN polypeptide having the amino acid sequence of SEQ ID        NO: 4; or    -   (v) an active fragment of any one of (i)-(iv) having at least        100, 110, 120 or 130 amino acids, wherein said PTN polypeptide        of any one of (i)-(iv) or said active fragment of (v) is capable        of binding to its specific receptors, such as RPTPβ/ζ, ALK or        Syndecan 3 polypeptide.

In certain cases of the transgenic animal according to the inventionsaid promoter is a brain-specific promoter. Preferably, the promoter isspecific for neurons. In certain cases, in accordance with this andother aspects of the present invention, the promoter is selected fromthe group consisting of: the human or mouse Thy1 gene promoter; human ormouse Neuron specific enolase (NSE); human or mouse Rhombotin I; PGK;human or mouse Neurofilament Low (NF-L); human or mouse dopaminebeta-hydroxylase (DBH); human or mouse Synapsin-1. Further details ofthe promoters are provided in Table 4 below. A particularly preferredpromoter is the promoter of the Thy1 gene, especially a Thy1 promoter ofthe same species as the transgenic animal. The Thy1 promoter maycomprise or consist of a polynucleotide having at least 80%, 90%, 95% or99% nucleotide sequence identity to the sequence of SEQ ID NO: 5. Thepresent inventors have found that the Thy1 promoter permits elevatedexpression of PTN in a targeted manner; expression is largely confinedto cortical and hippocampal neurons, and also in other regions as theamygdala. Thus, use of the Thy1 promoter to drive expression of PTNprovides an advantageous way to induce the desired anxious and/ordepressive-like phenotype of certain embodiments of the transgenicanimal of the invention.

The transgenic animal of the invention has greater than wild-typeexpression of the PTN polypeptide, as defined herein, in at least onebrain region selected from: cortex and hippocampus. Results disclosedherein indicate that elevated expression of PTN in one or more of thesebrain regions contributes to, or underlies the anxious-depressive-likephenotype observed. The elevated expression may, in some cases, be arelatively modest increase in expression compared with wild-type (e.g.compared with the expression in the same brain region of a species-,gender- and age-matched wild-type animal which does not carry anygenetic alteration relating to PTN or its promoter). In some cases,elevated expression may be at least 10%, 20%, 30%, 50%, 100%, 200%, 300%or 400% or greater expression of the PTN polypeptide in said at leastone brain region. A variety of techniques are available for measuringexpression of PTN, including techniques for direct measurement ofprotein levels (e.g. Western blot and immunofluorescence) and techniquesfor indirect measurement based on measurement of mRNA encoding the PTNpolypeptide (e.g. qPCR).

The transgenic animal of the invention is preferably a rodent, such as amouse or rat. In some cases the transgenic animal of the invention maybe a non-human primate or other laboratory animal such as a dog or cat.Most preferably, the transgenic animal is a mouse (e.g. Mus musculus).

In a second aspect the present invention provides a vector comprising apolynucleotide encoding a PTN polypeptide operably linked to abrain-specific promoter which is other than a ptn gene promoter andother than the human PDGF beta-chain gene promoter and, optionally,further regulatory sequences. Preferably, said polynucleotide encodes aPTN polypeptide which is a mouse, rat or human PTN polypeptide, avariant (such as a splice variant), precursor (such as the 168 aminoacid human pleiotrophin precursor sequence available at NCBI accessionnumber NP_002816; GI: 4506281 or the 168 amino acid mouse pleiotrophinprecursor sequence available at NCBI accession number NP_032999; GI:6679543), derivative (such as a post-translationally processedpolypeptide), homologue or orthologue from another species (preferably amammalian homologue or orthologue) or fragment. The PTN polypeptide orfragment thereof preferably exhibits biological activity, particularlythe ability to bind to its specific receptors RPTPβ/ζ, ALK or Syndecan3. In preferred cases of the vector of this aspect of the invention saidpolynucleotide encodes:

-   -   (i) a PTN polypeptide having an amino acid sequence having at        least 80%, 90%, 95% or 99% amino acid sequence identity to the        sequence of SEQ ID NO: 2;    -   (ii) a PTN polypeptide having the amino acid sequence of SEQ ID        NO: 2    -   (iii) a PTN polypeptide having an amino acid sequence having at        least 80%, 90%, 95% or 99% amino acid sequence identity to the        sequence of SEQ ID NO: 4;    -   (iv) a PTN polypeptide having the amino acid sequence of SEQ ID        NO: 4; or    -   (v) an active fragment of any one of (i)-(iv) having at least        100, 110, 120 or 130 amino acids, and wherein said PTN        polypeptide of any one of (i)-(iv) or said active fragment        of (v) is capable of binding to specific receptors RPTPβ/ζ, ALK        or Syndecan 3 polypeptide.

Said promoter of the vector of this aspect of the invention may bespecific or largely specific to neurons. A preferred promoter of thevector of this aspect of the invention is a Thy1 promoter. The Thy1promoter may comprise or consist of a polynucleotide having at least80%, 90%, 95% or 99% nucleotide sequence identity to the sequence of SEQID NO: 5. The vector of this aspect of the invention may find use in thepreparation of a transgenic animal model of the first aspect of theinvention.

In a third aspect the present invention provides an in vitro method foridentifying an agent (e.g. a small molecule, a nucleic acid or aprotein) for use in the treatment of neuropsychiatric illnesses,particularly mood disorders such as anxiety and/or major depression,comprising:

-   -   (i) contacting a cell that expresses a PTN polypeptide with a        test agent and measuring, directly or indirectly, expression of        the PTN polypeptide relative to expression of the PTN        polypeptide in a control cell which has not been exposed to the        test agent; and/or    -   (ii) contacting a PTN polypeptide with a test agent and        measuring, directly or indirectly, binding of the PTN        polypeptide to specific receptors RPTPβ/ζ, ALK or Syndecan 3        relative to the binding of a control PTN polypeptide which has        not been exposed to the test agent to specific receptors        RPTPβ/ζ, ALK or Syndecan 3 polypeptide; and/or    -   (iii) contacting a cell that presents PTN specific receptors        with a PTN polypeptide and with a test agent and measuring,        directly or indirectly, neurogenesis (proliferation,        differentiation or migration) and/or synaptogenesis.    -   wherein a reduction in said expression in (i) and/or a reduction        in said binding in (ii) and/or an alteration in neurogenesis        and/or synaptogenesis in (iii) due to the test agent indicates        that the test agent is potentially useful in the treatment of        neuropsychiatric illnesses, particularly mood disorders such as        anxiety and major depression. Preferably, said cell that        expresses a PTN polypeptide and/or said cell that presents PTN        specific receptors is a neuronal cell or non-neuronal cell        obtained from:    -   a patient having a neuropsychiatric illness, particularly mood        disorders such as anxiety and/or major depression or,    -   a transgenic animal of the invention or,    -   a cell that has been transfected or transformed with a        polynucleotide encoding a PTN polypeptide or with a vector in        accordance with the second aspect of the invention or,    -   A cell that expresses at least one PTN specific receptor, such        as RPTPβ/ζ, ALK or Syndecan 3.

Expression of PTN polypeptide may be measured at any stage of expressionof the PTN-coding gene (e.g. measuring mRNA level or protein level). Inpreferred cases of the method of this aspect of the invention the testagent is found to reduce said expression in (i) and/or said binding in(ii) and/or neurogenesis and/or synaptogenesis in (iii). Such testagents may be regarded as functional antagonists of the PTN polypeptide(whether they act pre- or post-translationally). Test agents that arefound to reduce said expression in (i) and/or said binding in (ii)and/or neurogenesis and/or synaptogenesis in (iii) may be subjected tofurther screening (including in vivo screening as described furtherherein). In some cases the method of this aspect of the inventionfurther comprises isolating the test agent and, optionally, formulatingthe test agent into a pharmaceutical composition with at least onepharmaceutically acceptable salt, carrier or excipient.

In a related aspect the present invention provides an in vitro screeningmethod comprising:

-   -   contacting at least one cell that expresses a PTN polypeptide        and/or at least one PTN specific receptor, such as RPTPβ/ζ, ALK        or Syndecan 3 with a test agent; and    -   detecting whether said test agent alters a PTN-related activity        as compared with said activity in the absence of the test agent.

The method may comprise detecting a change in PTN-related activity inthe presence of the test agent as compared with said PTN-relatedactivity in the absence of the test agent. The method may comprisecomparing the PTN-related activity of a cell exposed to the test agentwith said PTN-related activity of a (second) “control cell” which hasnot been exposed to the test agent. Additionally or alternatively, themethod may comprise comparing the PTN-related activity of a cell exposedto the test agent with said PTN-related activity of the same cell in theabsence of the test agent. For example a “baseline” of said PTN-relatedactivity may be established prior to addition of the test agent and thePTN-related activity assessed (e.g. relative to said baseline) afterexposing the cell to the test agent. Preferably, the cell is a cell thatexpresses at least one PTN specific receptor, or a cell that has beentransfected or transformed with a vector comprising a polynucleotidethat encodes said PTN polypeptide, e.g. a vector in accordance with thesecond aspect of the invention. In particular, the cell may be aneuronal cell line (e.g. a human or animal, such as rodent, derived cellline), and/or a primary culture that has been extracted from the brainof the transgenic mice of the present invention or from the brain of anyother animal or a stem cell-derived neuronal cell. The cell used in themethod according to this aspect of the invention may comprise a vectorcomprising a polynucleotide that encodes a PTN polypeptide operablylinked to a promoter, such as a promoter that permits variableexpression of the PTN. The promoter may be a Thy1 promoter as defined inaccordance with the tenth aspect of the invention. Alternatively oradditionally, a plurality of cells each comprising a vector comprising apolynucleotide that encodes a PTN polypeptide operably linked to apromoter may be used in accordance with the method of this aspect of theinvention. In some cases, the plurality of cells may comprise sub-setsof cells, wherein the cell of each sub-set has a vector having apromoter that differs from the promoter of the vectors of cells of otherof said sub-sets, such that the expression level of PTN differs betweensaid sub-sets of cells. Preferably, a first sub-set of cells ischaracterised by relatively low expression of PTN and a second sub-setof cells is characterised by higher expression of PTN relative to saidfirst sub-set of cells.

In a fourth aspect the present invention provides an in vivo method foridentifying an agent for use in the treatment of neuropsychiatricillnesses, particularly mood disorders such as anxiety and/or majordepression, comprising:

-   -   (i) administering a test agent to a transgenic animal of the        invention and subsequently measuring, directly or indirectly,        the expression of a PTN polypeptide in at least one brain region        relative to the expression of the PTN polypeptide in at least        one brain region of a control transgenic animal of the        invention, which has not been exposed to the test compound;        and/or    -   (ii) administering a test agent to a transgenic animal of the        invention and subsequently assessing the presence and/or        severity of one or more anxiety and/or depressive-related        behaviours in the transgenic animal relative to the one or more        depressive-related behaviours in a control transgenic animal of        the invention, which has not been exposed to the test agent,        and/or    -   (iii) administering a test agent to a transgenic animal of the        invention and subsequently evaluating the neurogenesis and        synaptogenesis processes in the transgenic animal relative to        the neurogenesis and synaptogenesis in a control transgenic        animal which has not been exposed to the test agent.

Wherein a reduction in said expression in (i) and/or said one or moreanxiety and/or depressive-related behaviours in (ii) and/or and increasein neurogenesis and/or synaptogenesis in (iii) due to the test agentindicates that the test agent is potentially useful in the treatment ofneuropsychiatric illnesses, particularly mood disorders such as anxietyand/or major depression. Said one or more anxiety and/ordepressive-related behaviours may be selected from: reduced motoractivity in an open-field test; reduced time spent in open arms of anelevated plus maze; reduced time in the lit portion of a light-dark box;increased latency to feed in a Novelty Suppressed Feeding test,increased time immobile on tail suspension test; and decreased sucroseintake . . . . Preferably, in the method of this aspect of the inventionthe test agent is found to reduce said expression in (i) and/or said oneor more anxiety and/or depressive-related behaviours in (ii) and/orincrease neurogenesis and/or synaptogenesis in (iii). Such test agentsmay be regarded as in vivo functional antagonists of PTN and/orPTN-associated anxiety, depressive-like behaviour, neurogenesis, and/orsynaptogenesis. In some cases the test agent is an agent which haspreviously been tested in a method of the third aspect of the invention.In this way an initial in vitro screen may be used to target subsequentin vivo screening on more promising candidate agents.

The method of this aspect of the invention may further compriseisolating the test agent and, optionally, formulating the test agentinto a pharmaceutical composition with at least one pharmaceuticallyacceptable salt, carrier or excipient.

In a fifth aspect the present invention provides an agent identified oridentifiable by a method of the fourth or fifth aspect of the invention.The agent may be for use in medicine. Preferably, the agent is for usein a method of treating a neuropsychiatric illness, particularly mooddisorders such as anxiety and/or major depression. Preferably, the agentcomprises:

-   -   an antibody molecule or binding fragment thereof capable of        binding to a PTN polypeptide (e.g. an PTN polypeptide as defined        in relation to any aspect of the present invention); or    -   an antisense nucleic acid, ribozyme, triple helix molecule,        siRNA, miRNA or other nucleic acid capable of inhibiting PTN        gene expression (e.g. which is capable of hybridising to at        least a portion of a polynucleotide that encodes an PTN        polypeptide as defined in relation to any aspect of the present        invention or which is capable of hybridising to at least a        portion of a polynucleotide which is complementary to the        polypeptide that encodes the PTN polypeptide).    -   a small molecule, antibody molecule or binding fragment,        antisense nucleic acid, ribozyme, triple helix molecule, siRNA,        miRNA capable of antagonising the effect of PTN over-expression.

In a sixth aspect the present invention provides use of an agentidentified or identifiable by a method of the fourth or fifth aspect ofthe invention in the preparation of a medicament for treatment ofneuropsychiatric illnesses, particularly mood disorders such as anxietyand/or major depression. Said agent may be as defined in relation to thesixth aspect of the invention.

In an seventh aspect the present invention provides a method fortreating a neuropsychiatric illness, particularly mood disorders asanxiety and/or major depression, in a subject (e.g. a human patient inneed of said treatment), comprising administering a therapeuticallyeffective amount of an agent identified or identifiable by a method ofthe fourth or fifth aspect of the invention. Said agent may be asdefined in relation to the sixth aspect of the invention.

In an eighth aspect the present invention provides a method of assessingthe presence of or susceptibility to a neuropsychiatric illness in asubject, particularly mood disorders such as anxiety and/or majordepression, comprising:

-   -   detecting and/or determining the amount of a PTN polypeptide        and/or the amount of an mRNA or cDNA encoding an PTN polypeptide        in a sample which has been obtained from said test subject; and    -   comparing said amount of the PTN polypeptide and/or said amount        of the mRNA or cDNA encoding the PTN polypeptide with one or        more reference values corresponding to the amount of the PTN        polypeptide and/or the amount of the mRNA or cDNA encoding the        PTN polypeptide in a control sample obtained from a control        subject not having a neuropsychiatric illness. The sample may        comprise blood, plasma, serum, cerebrospinal fluid (CSF) or        tissue. Preferably, the sample comprises central nervous system        tissue (e.g. prefrontal cortex tissue).

In certain cases of the method of this aspect of the invention the testsubject has not previously been diagnosed as having a neuropsychiatricillness, such as anxiety and/or major depression. In certain other casesof the method of this aspect of the invention the test subject haspreviously been diagnosed as having a neuropsychiatric illness. Themethod of this aspect of the invention may be used to assess the stageand/or severity of the neuropsychiatric illness, to select the patientsthat will benefit from the treatments, or to monitor the effect of atreatment administered to the test subject.

In a ninth aspect the present invention provides a vector comprising apolynucleotide encoding a psychiatry-associated polypeptide operablylinked to a Thy1 promoter and optionally further regulatory sequences.Preferably, the Thy1 promoter comprises or consists of a polynucleotidehaving at least 80%, 90%, 95% or 99% nucleic acid sequence identity tothe sequence of SEQ ID NO: 5 or the Thy1 promoter comprises or consistsof a polynucleotide having the sequence of SEQ ID NO: 5. Thepolynucleotide encoding a psychiatry-associated polypeptide ispreferably a gene, the elevated expression of which has been found to beassociated with a neuropsychiatric illness (e.g. mood disorders such asanxiety and/or major depression). In accordance with this aspect of theinvention, the psychiatry-associated polypeptide is preferably a geneproduct of a gene set forth in Table 1 or Table 3 herein.

In a tenth aspect, the present invention provides use of a vector of theninth aspect of the invention, in the production of a transgenic animalhaving greater than wild-type expression of said psychiatry-associatedpolypeptide in at least one brain region. The transgenic animal soproduced and its offspring may be utilised in screening of test agentsfor potential treatments of the psychiatric disorder, such as anxietyand/or major depression.

The present invention includes the combination of the aspects andpreferred features described except where such a combination is clearlyimpermissible or is stated to be expressly avoided. Section headingsused herein are for convenience only and are not to be construed aslimiting in any way. These and further aspects and embodiments of theinvention are described in further detail below and with reference tothe accompanying examples and figures.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B provide information regarding samples included in thestudy. In FIG. 1 the information associated with each sample isdistributed in columns: FIG. 1A) data for major depressive subjects andFIG. 1B) data of the respective matched control subjects. The first ofthese corresponds to the sample code, and is followed by the sex, age,post mortem delay (PMD) and finally the toxicological data (drug freecorresponds to negative toxicology). The table also includes in whichkind of experiment the samples were used: chips from Affymetrix,validation of candidate genes by RT-PCR and characterization of proteinslevels by western blot.

FIGS. 2A-2C are a series of panels showing quantification of PTN intotal homogenate of human brain. FIG. 2A) Immunoreactivity for the PTNprotein determined in total homogenate of the prefrontal cortex(Brodmann area 9) of depressive subjects who died by suicide (n=22) andseparated into untreated (drug-free; DF, n=10) and treated withantidepressant drugs (treated; T, n=12). The data are expressed as apercentage of the mean value±SEM (standard error). The relative contentof each protein was calculated as percent change in relation to in-geltriplicate standards fixed at 100%, (pool of controls samples). Thetotal group (All) of depressive subjects (123±9%, n=22, **p<0.009,one-sample t-test) and the group of untreated depressive subjects(135±14%, n=10, **p=0.006, one-sample t-test) give significantly highervalues than the controls. However, the group of treated subjects(112±10%, n=12, p=0.2, not significant, one-sample t-test) give nosignificantly higher values than controls. FIG. 2B) Immunoreactivity forthe actin protein determined in total homogenate of the prefrontalcortex (Brodmann area 9) of depressive subjects who died by suicide(n=22) and separated into untreated (drug-free; DF, n=10) and treatedwith antidepressant drugs (treated; T, n=12). The data are expressed asa percentage of the mean value±SEM (standard error). The relativecontent of each protein was calculated as percent change in relation toin-gel triplicate standards fixed at 100%, (pool of controls samples).The total group (All) of depressive subjects (99±3%, n=22, notsignificant), the group of untreated depressive subjects (96±4%, n=10,not significant) and the group of treated subjects (101±4%, n=12, notsignificant,) give similar values than controls. FIG. 2C) Representativeautoradiograms (“immunoblots”) of the protein PTN in post-mortem humanbrain (prefrontal cortex, Brodmann area 9) of untreated depressivesubjects who died by suicide (drug-free; DF), those treated withantidepressant drugs (treated; T) and their respective controls (C).Brain samples containing 40 μg of total protein each were loaded onto10% polyacrylamide gels. [C1=♀, 35 years, 17 hours PMD; DF1=♀, 35 years,23 hours PMD; C2=♀, 57 years, 4 hours PMD; DF2=♀, 54 years, 12 hoursPMD; where PMD is the time between death and autopsy].

FIGS. 3A-3F are a series of panels regarding generation of miceoverexpressing PTN. FIG. 3A) The transgene construct. FIG. 3B) Southernblot showing expression of PTN in transgene-negative (WT: lanes 3,4,6,7y 8) and transgene-positive (PTN-overexpressing “PTN-OE”: lanes 5 and 9)littermates (Ctr: endogenous mRNA; M: marker; WT: Transgene-negative;TG: mRNA transcribed from the transgene) using the complete PTN cDNA asa probe. FIG. 3C) agarose gel microphotography following PCRamplification (WT: wild type mouse; Tg: transgenic mouse). FIG. 3D) PTNmRNA hippocampal and cerebral cortex expression levels in PTN-OE malemice and WT littermates. Values are shown as relative expression level2^(ΔΔ)CT. FIG. 3E) Immunoreactivity for the PTN protein determined intotal homogenate of hippocampus and cerebral cortex (respectively) ofwild type control mice and transgenic mice (PTN-OE). Data are expressedas a percentage of the mean value±SEM (standard error) and expressed aspercentage of control group. **p<0.01, *p<0.04 when compared with thecorresponding control group (the two-tailed one-sample t-test). FIG. 3F)Neuroanatomical distribution of PTN mRNA in WT and PTN-OE mice.

FIG. 4.—Semi-quantification of PTN mRNA levels in several areas inwild-type (wt) and transgenic (tg) animals. Relative optical densitiesare shown. aCA1, anterior CA1 hippocampal field; aCA3, anterior CA3hippocampal field; aDG, anterior dentate gyrus; aCx, anterior cingulatedcortex; aCPu, anterior caudate-putamen; aAmy, anterior amygdale nuclei;aRt, anterior Reticular thalamic nuclei; mCA1, medial CA1 hippocampalfield; mCA3, medial CA3 hippocampal field; mDG, medial dentate gyrus;mCx, medial cingulated cortex; mDPu, medial caudate-putamen; mRt, medialReticular thalamic nuclei; pCA1, posterior CA1 hippocampal field; pCA3;posterior CA3 hippocampal field; pDG, posterior dentate gyrus; pCx,posterior cingulated cortex; PK, Purkinje cells of the cerebellum; GR,granular cells of the cerebellum; nd: no determined.

FIGS. 5A-5D illustrate immunohistochemistry experiments for PTN inhippocampus (FIGS. 5A and 5B) and cerebral cortex region (FIGS. 5C and5D) of the brain in control (FIGS. 5A and 5C) and in transgenic mice(FIGS. 5B and 5D).

FIG. 6.—General observation of animal behavior. Results are expressed as% of PTN-OE and Wild-type animals exhibiting a given behavioralparameter of those described in Irwin's test protocol.

FIGS. 7A-7I are a series of panels showing assessment of spontaneouslocomotor activity and Anxiety-like behavior measured in the open fieldin controls and transgenic mice. The following parameters were measuredin the activity boxes: (FIG. 7A) total motor activity, (FIG. 7B)locomotor activity and (FIG. 7C) rearing. Data are expressed asmean±S.E.M (n=18, wild-type and n=21, transgenic). The followingparameters were measured in the open field under stressful conditions:(FIG. 7D) time spent in central area, (FIG. 7E) crossing squares incentral area, (FIG. 7F) entries in central area, (FIG. 7G) crossingsquares in peripheral area, (FIG. 7H) grooming and (FIG. 7I) rearing.Data are expressed as mean±S.E.M (n=18, wild-type and n=21, transgenic).*p<0.05 and **p<0.01 when comparing with wild-type group.

FIGS. 8A-8I are a series of panels showing assessment of anxiety-likeand depressive-like behaviours, Tail Suspension Test and in SucroseIntake in transgenic and wild-type mice. Elevated Plus Maze: FIG. 8A)Number of entries in open arms; FIG. 8B) Percent of distance that micespent in the open arms; FIG. 8C) Percent of time that mice remain in theopen arms (Behaviour was evaluated for a period of 5 min). Light-DarkBox: FIG. 8D) Latency to go for the first time to the lit compartment;FIG. 8E) time spent in the lit compartment; FIG. 8F) Number of squarescrossed FIG. 8G) number of visits into each zone of the lit compartmentare recorded for 5 min through a videocamera system. Tail suspensiontest: FIG. 8H) Time of immobility during 6 minutes session; Sucroseintake Test: FIG. 8I) Performance in the sucrose intake test show areduced intake of sucrose

Data are expressed as mean±SEM of percentage. Values from PTN-OE mice(black columns) that are significantly different (*p<0.05, **p<0.002 thetwo-tailed one-sample t-test) from control mice (white columns) areindicated.

FIG. 9.—LTP induced in hippocampal slices of transgenic PTN mice.Attenuated LTP in mice overexpressing PTN. The symbols represent fEPSPslope values in hippocampal slices from CA1. After a basal 20 minperiod, 3 trains of HFS (indicated by arrow) were applied in PTNtransgenic mice (•, n=8) and control slices (∘, n=6).

FIG. 10.—Shows the mouse (Mus musculus) PTN cDNA sequence availableunder NCBI Accession No BC061695.1 [GI:38197284] (SEQ ID NO: 1).

FIG. 11.—shows the mouse (Mus musculus) PTN predicted translatedsequence. The mature form of the protein is residues 33-168 availableunder NCBI Accession No. NM_008973 [GI:118130571] (SEQ ID NO 2).

FIG. 12.—Shows the human Pleiotrophin (PTN) cDNA sequence availableunder NCBI Accession No. BC005916.1 [GI:13543514] (SEQ ID NO: 3).

FIG. 13.—Shows the human PTN amino acid sequence available under UniprotAccession No. P21246, version 1, 1 May 1991. The mature sequence isresidues 33-168 (SEQ ID NO: 4).

FIGS. 14A-D are a series of panels showing the sequence of the acceptorvector pTSC-a2 which contained the regulatory regions responsible fortissue specific expression of the mouse (Mus musculus) THYmocytedifferentiation antigen 1 gene (Thy-1) sequence (SEQ ID NO: 5).

FIG. 15. —Anxiety-like responses and sensitivity to chronic Fluoxetinetreatment assessment by Novelty Suppressed Feeding Test. Latency to feed(in seconds) is shown on the y-axis for wild-type (WT; open bars) andtransgenic (TG; black bars) mice, for untreated (−Flx) and treated withfluoxetine (+Flx); (n=14, wild-type and n=14, transgenic); ***p<0.0002.

FIG. 16.—Neurogenesis. Number of bromodeoxyuridine (BrdU) positive cellsin wild-type (WT) and transgenic (TG) mice.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Jun. 20, 2016,and is 23,221 bytes, which is incorporated by reference herein.

DETAILED DESCRIPTION OF THE INVENTION

The terms “subject” or “individual” refer to members of mammalian animalspecies and include, but are not limited to, domestic animals, primatesand humans; the subject is preferably a male or female human being ofany age or race.

The term “neuropsychiatric illness” includes a wide range of undesirablepsychiatric and neurological conditions, such as schizophrenia, bipolardisorder, anxiety, major depression, schizoaffective disorder,psychiatric conditions (defined in the DMS IV manual) and neurologicalillnesses caused by alterations of the central nervous system.

The term “gene” refers to a region of a molecular chain ofdeoxyribonucleotides that encodes a protein and which could representthe complete coding sequence or a portion of it.

The term “DNA” refers to deoxyribonucleic acid. A DNA sequence is asequence of deoxyribonucleotides.

The term “RNA” refers to ribonucleic acid. An RNA sequence is a sequenceof ribonucleotides.

The term “mRNA” refers to messenger ribonucleic acid, which is thefraction of total RNA that is translated into proteins.

The term “cDNA” refers to a sequence of nucleotides that iscomplementary to an mRNA sequence.

The phrase “mRNA transcribed from” refers to the transcription of thegene (DNA) into mRNA, as the first step for the gene to be expressed andtranslated into protein.

The term “nucleotide sequence” refers equally to a sequence ofribonucleotides (RNA) or deoxyribonucleotides (DNA).

The term “protein” refers to a molecular chain of amino acids joined bycovalent or non-covalent bonds. This term includes all types ofpost-translational modification, such as glycosylation, phosphorylationor acetylation.

The terms “peptide” and “polypeptide” refer to molecular chains of aminoacids that represent a protein fragment. The terms “protein” and“peptide” are used indistinctly.

The term “antibody” refers to a glycoprotein that displays specificbinding to a target molecule, which is termed the “antigen”. The term“antibody” includes monoclonal antibodies or polyclonal antisera, eitherintact or fragments thereof; it includes human, humanised and non-humanantibodies. “Monoclonal antibodies” are homogeneous populations ofhighly specific antibodies that target a unique antigenic site or“determinant”. “Polyclonal antisera” include heterogeneous populationsof antibodies that target different antigenic determinants.

The term “epitope”, as used in the present invention, refers to anantigenic determinant of a protein, which is the amino acid sequence ofthe protein that a specific antibody recognises.

The term “solid phase”, as used in the present invention, refers to anon-aqueous matrix to which an antibody can be bound. Examples of solidphase materials include glass, polysaccharides such as agarose,polyacrylamide, polystyrene, polyvinyl alcohol and silicones. Examplesof solid phase forms are the well of a test plate or a purificationcolumn.

The terms “oligonucleotide” and “oligonucleotide primer” are usedindistinctly and as used in the present invention, refer to nucleotidesequences that are complementary to a nucleotide sequence in the ptngene. Each primer hybridises with its target nucleotide sequence andacts as a starting point for nucleotide polymerisation catalysed by DNApolymerase, RNA polymerase or reverse transcriptase.

The term “probe”, as used in the present invention, refers to anucleotide sequence that is complementary to a nucleotide sequencederived from the ptn gene and which can be used to detect thisnucleotide sequence derived from the ptn gene.

The term “therapeutic target” refers to nucleotide or peptide sequencesagainst which a drug or therapeutic compound can be designed and appliedclinically.

The term “antagonist” refers to any molecule that inhibits thebiological activity of the antagonised molecule. Examples of antagonistsinclude, amongst others, proteins, peptides, sequence variations ofnatural peptides and small organic molecules (molecular weights of lessthan 500 Daltons).

The term “exogenous promoter” as used herein means a promoter other thanthe PTN gene promoter.

The term “to exhibit depressive symptoms” means, but is by no meanslimited to, particularly to exhibit an increase in immobility time inthe tail suspension test as described below.

The term “to exhibit anxious symptoms” means, but is by no means limitedto, particularly to exhibit a reduction in time spent in the open armsin the elevated plus maze as described below.

The term “animal model of mood disorders” means an animal which may beused in detecting an effect of a test substance on the treatment foranxiety and/or depression or screening for an agent for treating anxietyand/or depression.

PTN

As used herein PTN polypeptide may be a native PTN polypeptide from amammalian species, particularly a mouse, human or rat. PTN protein isalso known by the names: HBBM (Heparin-binding brain mitogen), HBGF-8(Heparin-binding growth factor 8), HB-GAM (Heparin-bindinggrowth-associated molecule), HBNF-1 (Heparin-binding neuriteoutgrowth-promoting factor 1) and OSF-1 (Osteoblast-specific factor 1).Also encompassed by the term PTN polypeptide as used herein are variants(such as a splice variant), precursors (such as the 168 amino acid humanpleiotrophin precursor sequence available at NCBI accession numberNP_002816; GI: 4506281 or the 168 amino acid mouse pleiotrophinprecursor sequence available at NCBI accession number NP_032999; GI:6679543), derivatives (such as a post-translationally processedpolypeptide) and fragments thereof. The PTN polypeptide or fragmentthereof preferably exhibits biological activity, particularly theability to bind to a cell surface receptor such as Protein TyrosinePhosphatase beta/zeta (RPTP β/ζ), Anaplastic Lymphoma Kinase Receptor(ALK) or N-Syndecan (SDC3) (for example a cell surface receptor of thesame species). The PTN polypeptide may comprise:

-   -   (i) a PTN polypeptide having an amino acid sequence having at        least 80%, 90%, 95% or 99% amino acid sequence identity to the        sequence of SEQ ID NO: 2;    -   (ii) a PTN polypeptide having the amino acid sequence of SEQ ID        NO: 2;    -   (iii) a PTN polypeptide having an amino acid sequence having at        least 80%, 90%, 95% or 99% amino acid sequence identity to the        sequence of SEQ ID NO: 4;    -   (iv) a PTN polypeptide having the amino acid sequence of SEQ ID        NO: 4; or    -   (v) an active fragment of any one of (i)-(iv) having at least        100, 110, 120 or 130 amino acids, wherein said PTN polypeptide        of any one of (i)-(iv) or said active fragment of (v) is capable        of binding to a cell surface receptor such as Protein Tyrosine        Phosphatase beta/zeta (RPTP β/ζ), Anaplastic Lymphoma Kinase        Receptor (ALK) or, N-Syndecan (SDC3) (for example a cell surface        receptor of the same species).

As used herein a PTN-encoding polynucleotide or similar expressionrefers to any nucleic acid (DNA or RNA) that encodes a PTN polypeptideas defined herein. Preferred PTN-encoding polynucleotides include thosehaving at least 80%, 90%, 95% or 99% nucleotide sequence identity to thepolynucleotide sequence of SEQ ID NO: 1 or 3. Particularly preferredPTN-encoding polynucleotides comprise or consist of a polynucleotidehaving the polynucleotide sequence of SEQ ID NO: 1 or 3.

Thy1 Promoter

The nucleotide sequence of the mouse wild-type Thy1 gene is shown inFIG. 12 (SEQ ID NO: 5). As used herein the Thy promoter may be a variantor homologue from a non-mouse species, wherein said variant or homologuecomprises or consists of a polynucleotide sequence having at least 80%,90%, 95% or 99% sequence identity to the polynucleotide sequence of SEQID NO: 5, or a fragment thereof having promoter activity. Preferably,the Thy1 promoter comprises or consists of a polynucleotide sequencehaving the polynucleotide sequence of SEQ ID NO: 5.

Neuro-Specific Promoters

Other Neuro-specific promoters (i.e. other than the Thy1 promoter) thatfind use in accordance with the present invention, for example asalternatives to the Thy1 promoter, include: human or mouse Neuronspecific enolase (NSE or ENO2); human or mouse Rhombotin I (LMO1); humanor mouse Phosphoglycerate kinase 1 (PGK or PGK11); human or mouseNeurofilament, light polypeptide (NF-L); human or mouse dopaminebeta-hydroxylase (DBH); human or mouse Synapsin-1 (SYN1). Furtherdetails concerning the neuro-specific promoters are provided in Table 4.In particular, the skilled person is able to determine suitable promotersequence from the genomic reference sequence details provided in Table 4(e.g. making use of one or more promoter/regulatory sequence elementsfrom the 5′ 10 kb or 5′ 100 kb genomic sequence as identified in columns7 and 8, respectively, of Table 4.

Transgenic Animals

The non-human transgenic animal of the invention is preferably a rodent,most preferably a mouse. A variety of suitable techniques may be used toalter the mouse genome to enhance PTN expression in at least one brainregion. Preferably, a vector of the invention is introduced into anon-human embryo. The incorporated polynucleotide encoding a PTNpolypeptide and under control of a promoter is preferably transmissiblebetween generations. This facilitates establishment of a colony oftransgenic animals. Preferably, genomic incorporation of thepolynucleotide is verified by extraction and characterisation of DNAfrom the transgenic animal and/or its offspring.

Screening Methods and Test Agents

When an agent reduces the expression levels of the ptn gene or reversesthe effects due to increased expression of said gene, this agent becomesa candidate for the treatment of neuropsychiatric disorders.

Thus, the invention relates to the use of nucleotide or peptidesequences from the ptn gene in methods to search for, identify, developand assess the efficacy of compounds to treat neuropsychiatricillnesses, especially mood disorders such as anxiety and/or majordepression. The importance of screening methods in the search for drugsbased on the binding, competitive or otherwise, of a potential drugmolecule to the therapeutic target should be stressed.

Another object of the invention consists of providing agentscharacterised by their inhibition of the expression and/or activity ofthe PTN protein. Those agents which can be identified and assessedaccording to the present invention may be chosen from the group formedby:

a) a specific antibody, or combination of antibodies, against one ormore epitopes present in the protein PTN, preferably a human orhumanised monoclonal antibody, which can also include an antibodyfragment, a simple chain antibody or an anti-idiotype antibody;

b) cytotoxic agents, such as toxins, molecules containing radioactiveatoms, or chemotherapy agents, including but not limited to, smallorganic and inorganic molecules, peptides, phosphopeptides, antisensemolecules, ribozymes, siRNAs, triple helix molecules, etc., whichinhibit the expression and/or activity of the PTN protein; and

c) antagonists of the PTN protein that inhibit one or more functions ofsaid protein.

The present invention also provides a pharmaceutical composition thatcontains a therapeutically effective quantity of one or more agentsidentified in a screening method of the invention (in vitro or in vivomethod) together with one or more excipients and/or transportsubstances. Furthermore, said composition may comprise a further activeingredient that inhibits the function of the PTN protein.

The excipients, transport substances and auxiliary substances should bepharmaceutically and pharmacologically acceptable such that they can becombined with other components of the formulation or preparation andthey do not cause adverse effects on the treated organism. Thepharmaceutical compositions or formulations include those that areappropriate for oral or parenteral (including subcutaneous, intradermal,intramuscular and intravenous) administration, although the bestadministration route depends on the state of the patient. Theformulations can be in the form of simple doses and the formulations areprepared according to known methods in the field of pharmacology. Theamounts of active substances to be administered can vary depending onthe therapeutic needs.

Diagnostic Methods

Methods of the present invention for assessing the presence of orsusceptibility to a neuropsychiatric illness are based on theobservation that subjects or individuals diagnosed with neuropsychiatricillnesses, especially mood disorders, such as anxiety and/or depression,present much higher levels of the protein coded for the ptn gene (PTNprotein) than the corresponding levels in subjects with no clinicalhistory of these illnesses.

The method presented involves a subject sampling step, and can work withdifferent biological fluids such as, for example: blood, plasma, serumor cerebrospinal fluid. Preferably the sample comprises CNS tissue.

The samples can be taken from subjects previously diagnosed with a givenneuropsychiatric illness or from undiagnosed individuals, as well asfrom a subject receiving treatment or who has been treated previouslyfor a neuropsychiatric illness, particularly mood disorders such asanxiety and/or depression.

The present method may also involve an extraction step, either to obtainthe protein extract from the sample or to obtain the total RNA extract.

Any conventional in vitro test can be used for measurement of levels ofmRNA transcribed from the ptn gene or its complementary cDNA, or theconcentration of the PTN protein, in samples collected from theindividuals to be analysed and from control individuals.

Thus, in some cases the present invention provides an in vitro methodfor detecting the presence of neuropsychiatric illnesses in anindividual, especially mood disorders such as anxiety and/or depression,to determine the state or severity of this illness in the individual, orto monitor the effect of a treatment administered to an individual whopresents this illness, based either on measuring the concentration ofthe PTN protein or the expression of the ptn gene.

If the concentration of the PTN protein is to be determined, the methodmay comprise an initial step where the protein extract from the sampleis mixed with one or more specific antibodies against one or moreepitopes of the PTN protein, and a second step where the complexesformed between these antibodies and the protein PTN are quantified.

A wide variety of immunological tests can be used to detect theformation of specific antigen-antibody complexes and several competitiveand non-competitive protein binding assays have been describedpreviously, a large number of which are available commercially.

Thus, the PTN protein can be quantified with antibodies such as specificmonoclonal and polyclonal antibodies, either intact or recombinantfragments thereof, combibodies, and Fab or scFv antibody fragmentsagainst the PTN protein. These antibodies can be human, humanised ornon-human in origin. The antibodies used in these tests can be labelledor not and the unlabelled antibodies can be used in clumping tests whilethe marked antibodies can be used in a wide range of tests. Thelabelling molecules that can be used to label the antibodies includeradionuclides, enzymes, fluorophores, chemoluminescent reagents,enzymatic substrates or cofactors, enzyme inhibitors, particles,colorants and derivatives.

A wide range of well-known tests that use unlabelled (primary antibody)and labelled antibodies (secondary antibody) can be used with theinvention dealt with here. These techniques include the Western blot orWestern transfer, ELISA (Enzyme-Linked inmunosorbent assay), RIA(Radioimmunoassay), competitive EIA (competitive enzyme immunoassay),DAS-ELISA (double antibody sandwich-ELISA), immunocytochemical andimmunohistochemical techniques, techniques based on the use of biochipsor protein microarrays that include specific antibodies or tests basedon colloidal precipitation in formats such as dipsticks. Other methodsof detecting and quantifying the PTN protein include affinitychromatography techniques, ligand binding tests, mass spectrometry orlectin binding tests.

If the mRNA or cDNA corresponding to the ptn gene is to be detected inaddition to or as an alternative to detecting the protein, the method ofthe invention may comprise extraction of RNA (such as total RNA). ThemRNA or cDNA corresponding to the ptn gene is detected by amplifying thetotal RNA extract or the corresponding cDNA synthesised by reversetranscription from the mRNA template in a first step, followed by asecond step that involves quantification of the product amplified fromthe mRNA or cDNA from the ptn gene.

An example of mRNA amplification consists of reverse transcribing themRNA into cDNA (RT), and then performing a polymerase chain reaction(PCR) with primer oligonucleotides. PCR is a technique that is used toamplify a certain nucleotide sequence (target) contained in a mixture ofnucleotide sequences. PCR uses an excess of a pair of primeroligonucleotides that hybridise with the complementary strands of thetarget nucleotide sequence. Next, an enzyme with polymerase activity(Taq DNA polymerase) extends each primer by using the target nucleotidesequence as a template. The extension products are then converted intotarget sequences upon dissociation of the original target strand. Newprimer molecules then hybridise to them and the polymerase extends them.This cycle is repeated to increase the number of target sequencesexponentially and it is a technique described in the U.S. Pat. No.4,683,195 and U.S. Pat. No. 4,683,202. Many methods for detecting andquantifying the products of PCR amplification, any of which can be usedin this invention, have been described previously. In a preferred methodof the invention, the amplified product is detected by agarose gelelectrophoresis as follows: five microlitres of the amplificationproduct is separated by electrophoresis on a 2% agarose gel in a TBE0.5× buffer at 100 vdc for one hour. After electrophoresis, the gel isstained with ethidium bromide and the amplification product visualisedby illuminating the gel with ultraviolet (uv) light. As an alternativeto staining and also as a preferred technique, the amplification productcan be transferred to a nylon membrane by the Southern blottingtechnique and detected with a specific, appropriately labelled probe forthe cDNA of the ptn gene.

In another example the mRNA is detected by transferring the mRNA to anylon membrane by transfer techniques such as Northern blot anddetection with specific probes for the mRNA or the corresponding cDNAfor the ptn gene. In another specific assay, the mRNA corresponding tothe ptn gene may be amplified and quantified at the same time byreal-time quantitative RT-PCR (Q-PCR).

The method may involve comparing the amount of the PTN protein, theamount of mRNA from the ptn gene or the amount of the corresponding cDNAdetected in the sample taken from the subject with the amount of PTNprotein, the amount of mRNA from the ptn gene or the amount of thecorresponding cDNA detected in samples from one or more control subjectsor with one or more pre-determined reference values. An increase ofaround 10%, preferably 20%, 30%, 50% or greater may indicate thepresence of or susceptibility to a neuropsychiatric illness in thesubject.

The following is presented by way of example and is not to be construedas a limitation to the scope of the claims.

EXAMPLES Example 1 Analysis of Brain Tissue Using Microarray DataAnalysis

Samples of post-mortem prefrontal cortex (Broadmann's Area 9) wereobtained at autopsies performed in the Forensic Anatomical Institute,Bilbao, Spain, and stored at −80° C. The study was developed incompliance with policies of research and ethical review boards forpost-mortem brain studies (Basque Institute of Legal Medicine, Bilbao).Deaths recorded as suicide were subjected to retrospective carefulsearching for previous medical diagnosis and treatment using examiner'sinformation and records of hospitals and mental health centres.Depressed victims were selected according to the following criteria:suicide by violent method, lifetime diagnosis of Major Depression madeaccording to DSM-IV and DSM-IV-R (American Psychiatric Association.Diagnostic and Statistical Manual of Mental Disorders, 4rd editionrevised. American Psychiatric Association, Washington DC, 2000), absenceof psychotropic or illegal drugs. Matched controls did not have apsychiatric disorder, were selected on the basis of sex, age andpost-mortem delay (PMD) and suffered a sudden and unexpected death. Incontrol subjects other drug exposures were minimal (benzodiazepine,salicylic acid, antiarrythmic, diuretics). The groups did not differstatistically for mean age or PMD. A summary of the demographicinformation of subjects used in this study is shown in FIG. 1.

Total RNA was isolated from brain tissue with TRIZOL Reagent (InvitrogenCo.) as directed by the manufacturer. RNA quality and integrity wereverified by measuring the A_(260/280) ratio and on agarose gels.Purified RNA was cleaned with RNeasy Minikit (QIAGEN Inc.), and 10 μg oftotal RNA were used for cDNA synthesis using SuperScript™ II RNase(Invitrogen Co.). Biotinylated cRNA was synthesised using ENZO BioArray™HighYield™ Transcript Labeling Kit (Enzo Diagnostics). After in vitrotranscription, unincorporated nucleotides were eliminated using RNeasyMinikit columns (QIAGEN Inc.). Fifteen μg of biotinylated cRNA werehybridised to the Human Genome (HG_U133A) GeneChip® (Affymetrix Inc.)and automatically washed and stained using the Fluidics Station 400. Thechips were scanned using an Agilent GeneArray Scanner 2500 (AgilentTechnologies Inc.). All sample labelling, hybridisation, staining andscanning procedures were carried out using Affymetrix standardprotocols. A total of ten samples were individually hybridised to theGeneChips, five from each group. Exclusion criteria for microarrayhybridisation included male subjects below age 52 or above age 75, andany sample with PMD above 49 hours.

Scanned microarray data was processed using the algorithms in theMicroarray Analysis Suit Software MAS5.0 (Affymetrix). Global scalingwas performed to an arbitrary value of 100, and summarised data wasimported into GeneSpring 6.1 (Agilent Technologies Inc.). Normalisationprocedures were carried out following default options (measurements lessthan 0.01 were set to 0.01; per chip normalisation to 50^(th)percentile; per gene normalisation to median), and data were filtered inorder to remove unreliable measurements. A t-test (p<0.05) was conductedto obtain genes that showed a statistical difference of gene expressionbetween the two experimental groups. Fold Change (FC) values werecalculated after normalisation to the median of the intensity value ofthe control group. An arbitrary threshold value of 1 was chosen toselect upregulation or downregulation.

After filtering for inconsistent and unreliable data, the remaining10.906 probe sets were subjected to Analysis of Variance (ANOVA) todetermine genes that were differentially expressed. We defined a cutoffp-value of 0.05 to call a probe differentially expressed between the twosample groups studied. 226 probes passed this criterion, 82 of whichwere found to be upregulated in the experimental group compared to thecontrol group, and 144 were downregulated. Due to sample variability andthe small number of biological replicates per group, it was not possibleto correct for multiple testing. Nevertheless, we accepted being lessstringent at this point, in view of a validation of results using RT-PCRon a larger population. In addition to this, the MAS5.0 Change algorithmwas used to select a further set of genes that consistently had anIncrease or Decrease call across the different case-control pair wisecomparisons, including 62 upregulated sequences and 95 downregulatedsequences. Our cutoff criterion was rather relaxed for this analysis,due to variability issues. This method shared only one probe set withthe previous method, with no relevance to the biological data mining ofthe results. The final list of 383 selected genes is showed in tableAnnex 1.

Example 2 Determination of PTN as a Biological Marker for Depression

Microarray results were investigated using quantitative PCR. Eightythree genes were selected for quantitative PCR to validate their mRNAexpression on a larger sample set, by using Low Density Arrays (Annex2). Seven genes (RPL10, B2M, ENO2, ZFP207, ACTB, GAPDH and NEFH) wereselected to be used as reference genes. Validation was carried out usingLow Density Arrays (Applied Biosystems) in a total of 40 prefrontalcortex samples (20 case and 20 control samples). This tool allowed thesimultaneous interrogation of all the chosen target genes with tworeplicates for each gene using TaqMan® technology. Briefly, a 384-wellmicrofluidic card is loaded with the selected gene expression assays.Each card is composed of eight sample-loading ports, each connected to48 reaction wells. One microgram of RNA from each sample was reversedtranscribed using a high-capacity cDNA archive kit (Applied Biosystems)and random primers. 25 μl of cDNA are used for preparation of PCRmastermix. Samples are then pipetted into each port. After centrifuging,the card is placed on the 7900HT Fast Real Time PCR System (AppliedBiosystems). All protocols followed manufacturer's directions. Forsignal detection, the ABI Prism 7700 Sequence Detector System was used.The Ct value (cycle number at threshold) was used as a measure ofrelative amount of mRNA molecules. All measurements were produced induplicate, and the average of the two was taken for analysis. Duplicatesfor which the standard deviation was greater than 0.38 where eliminatedfrom the analysis. Data obtained were analysed using geNorm 3.3 software(Vandesompele et al., 2002). geNorm detects the most stable referencegenes from the set of tested housekeepings and calculates a geneexpression normalisation factor for each sample. The Ct values aretransformed to quantities using the ΔCt method and then normalised tothe average value of the control group for calculation of Fold Changevalues. Significant changes were assessed based on an unpairedtwo-tailed Student t-test on normalized ΔCt values after exporting datainto Microsoft Excel®. To account for possible confounding variables,like treatment and matching samples, different analyses were carried outapplying different sample exclusion criteria.

Statistical analyses of the Q-PCR results were carried out comparing thegroup of cases versus their matched controls. This analysis showed 54genes differentially expressed between cases and controls. The resultsof the statistical analysis are shown on Annex 3.

Example 3 Validation by Western Blotting of PTN as a Biological Markerfor Depression

The levels of PTN protein in human cerebral prefrontal cortex samples(Brodmann area 9) from individuals diagnosed with depression who died bysuicide (n=23) and who had (n=11) or who had not (n=12) receivedantidepressant treatment and control subjects with no history ofpsychiatric illness who died accidentally (n=23), were validated byWestern blotting with an antibody that specifically recognises saidprotein. The samples from depressive patients were paired with controlsamples on the basis of sex, age and post mortem delay. Target proteinwas quantified in pairs of subject with depression and the respectivematched control. The relative content of each protein was calculated(percent change) in relation to in-gel triplicate standards (100%, poolof controls samples). This quantification procedure was assessed 2-3times in different gels, and the resulting mean value of the targetprotein was used as a final estimate.

Human brain samples (100 mg) were homogenized in cold 50 mM Tris-HClbuffer, pH 7.5, containing various protease inhibitors (Sigma proteaseinhibitor cocktail 50 μl/g, antipain and chymostatin 10 μl/ml), andphosphatase inhibitors (1 mM cantharidin, 1 mM sodium fluoride, and 1 mMsodium orthovanadate). Then, a mix of detergents (10% igepal, 5% sodiumdeoxycholate, 1% sodium dodecyl sulfate (SDS), and CHAPS 250 mM) wasadded and samples were centrifuged at 14,000×g for 10 min. The pelletprotein content was determined with the BCA protein assay kit (Pierce,Madrid, Spain) using bovine serum albumin as a standard. Aliquots ofthis mixture were combined with solubilization loading buffer (25%glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.01% bromophenol blue, and 62.5mM Tris-HCl, pH 6.8), to reach a final concentration of 2 mg/ml. Themixture was denatures at 95° C. for 5 min, and stored at −80° C. untiluse.

In routine experiments, 40 μg proteins of each human or control humanpool brain sample was subjected to SDS-PAGE on 15% polyacrylamideminigels and proteins were then transferred to nitrocellulose membranes.Membranes were blocked for 1 h with Tris-buffered saline (TBS; 10 mMTris-HCl, pH 7.4, 140 mM NaCl) containing 3% skimmed milk, and thenincubated with one of the primary antibodies at 4° C. overnight (PBS-3%skimmed milk-0.1% Tween buffer). The following primary antibodies wereused: PTN (Goat polyclonal antibody anti-Pleiotrophin, productAF-252-PB, lot no. PW07, 1:500 dilution, R&D Systems); Mouse monoclonalantibody anti-β-actin (clone AC-15, lot No. 014K4840, Sigma ChemicalCo.) was used for immunodetection at a dilution of 1:5,000 of β-actinprotein as a control loading The nitrocellulose membranes were incubatedwith a fluorescent anti-Goat IgG secondary antibody (IRDye™ 800 CWRabbit, Rockland, Gilbertsville, Pa., USA) and an anti-Mouse IgGsecondary antibody (IRDye™ 800 CW Rabbit, Rockland, Gilbertsville, Pa.,USA) at a dilution of 1:3,000 and 1:5,000 respectively. Finally, themembranes were imaged using the LICOR Odyssey Image System. Duplicateproblem samples were evaluated and normalized as percentage of immunereactivity of the respective matched control (see FIG. 2).

A significant increase in the levels of PTN protein was observed inprefrontal cortex samples from depressive subjects when compared withthe levels of the same protein in control subjects with no clinicalhistory of psychiatric illness (123±8, n=22, p<0.02). Furthermore, itwas found that the expression of PTN protein was much higher in theprefrontal cortex of depressives who had died by suicide and who had notreceived pharmacological treatment when compared with samples from thesame brain region of control individuals with no history of psychiatricillness and who had died accidentally (135±14, n=10, p<0.03).Interestingly, it was found that the PTN levels in depressive subjectswho had been treated with antidepressant drugs but who had also died bysuicide were lower than those in untreated individuals and were similarto the control levels (112±10, n=12, not significant; FIG. 2).

Example 4 Method of Preparing Transgenic Animal

The polynucleotide to be introduced contains a promoter sequence capableof controlling the expression of the depression-related protein and, ifdesired, may further contain an enhancer sequence. Thedepression-related protein may be expressed specifically in the brain.The promoter for preparing the model animal in the present example wasspecifically chosen. This promoter is the Thy-1 (THYmocytedifferentiation antigen 1), also known as CD90, T25 or Thy1.2. The Thy-1gene is a neuronal gene specifically expressed in neurons. Therefore, adesired gene (in this case PTN) can be selectively expressed in thebrain, particularly in neurons, by using the promoter region of a Thy-1gene. The transgenic mice were found to exhibit anxiety and/ordepression symptoms as a result of overexpression of PTN in the brain.

A 1.6 kb EcoR1-XhoI fragment containing the entire coding sequence ofthe mouse PTN gen (SEQ ID NO: 1) was introduced downstream of the Thy-1promoter (SEQ ID NO: 5) at XhoI site in the pTSC-a2 plasmid, whichcontained the regulatory regions responsible for tissue specificexpression of Thy-1 gene. Transgenic mice were generated by standardpronucleus microinjection of the 5.8 kb fragment from the Thy1-PTNconstruct on a hybrid B6/SJL-F1J genetic background (FIG. 3a ). Ninetysix animals were born, 18 of them carrying the transgene insertion (12 ♂y 6 ♀), as analyzed by Southern-blot by using the complete PTN cDNA as aprobe. Two transgenic lines were chosen and all of the wild-typecontrols were littermates of the transgenic mice (L5 and L6) (FIG. 3b ).

Successful introduction of the gene may be determined by extracting DNAfrom a part of the body (for example the tail tip) and confirming thepresence of the introduced polynucleotide. Animals testing positive forthe introduced gene are regarded as founders. The introducedpolynucleotide is transmitted to 50% of the offspring, and it ispossible to efficiently obtain wild type and mutated animals (FIG. 3c ).

The transgenic animal prepared as described above and its offspringexhibiting anxiety and/or depression symptoms are useful in detecting atherapeutic effect on anxiety and/or depression and screening for atherapeutic agent or agents for anxiety and/or depression.

Example 5 PTN Expression Level Determination in the Brain of TransgenicMice: mRNA and Protein

—mRNA Quantification:

a) Q-RT-PCR: Total RNA was extracted with RiboPure™ kit (Ambio, LifeTechnologies Corporation) and its integrity (RNA integrity number, RIN)measured by the Caliper's RNA LabChip® kit using the Agilent 2100Bioanalyzer. 600 ng of this purified RNA were used to generate cDNAusing High Capacity cDNA Reverse Transcription kit (Applied Biosystem,USA). qRT-PCR was performed with ABI 7500 thermocycler (AppliedBiosystem, USA) following manufacturer's instructions, using TaqMan®Inventoried Gene Expression probes (PTN: Mm 00436062_m1; GAPDH: VIC/MGBProbe, AB 4353339E-0909023, Applied Biosystem Life Technologies). GAPDHwas used as house-keeping gene and its expression value was used tonormalize PTN expression values. For expression quantification Pfaffl's2^(−ΔΔ)CT method was used, assuming a 100% PCR efficiency.

The results showed a significant increase of PTN expression levels inthe hippocampus and cerebral cortex of transgenic mice of L5 compared totheir wild-type littermates (FIG. 3d ).

b) In situ hybridization: Three different oligonucleotides were designedand synthesized, complementary to different domains of the PTN mRNA:

Oligo 1:  (SEQ ID NO: 6)AGTGTCCACAGCTGCCAAGATGAAAATCAATGCAGGAAGGCAGCTGC; Oligo 2: (SEQ ID NO: 7) ACTCGGCGCCAGTGCGAGTGCCCTCCCGGGTGCCCAATCCACAGTCC;Oligo 3:  (SEQ ID NO: 8)AGGCTTGGGCTTGGTGAGCTTGCCACAGGGCTTGGAGATGGTGACAG.

They were labeled with ³³P-dATP and used separately to hybridize tissuesections. The 3 independent probes gave the same hybridization pattern(data not shown). We next used them to analyze the expression pattern ofthe PTN mRNA in the PTN transgenic mice and compared it with that ofwild-type brains. An example of four different brain levels in 1 wt and1 transgenic mice brains is shown in FIG. 3f , obtained from theautoradiograms. In general the expression can be seen in many neurons indifferent brain areas (see hippocampus and cerebral cortex), althoughsome expression in white matter areas cannot be excluded.

Autoradiograms for each hybridized probe were semiquantified with anMCID™ system and relative optical densities (ROD) were obtained.Measurements for PTN mRNA from 3 Wt and 3 TG mice are presented in FIG.4 where TG/WT ratio confirms PTN overexpression, mostly in hippocampalfields such as CA3 area (4-6-fold), CA1 and dentate gyrus (around2.5-fold).

—Protein quantification:

a) Western Blot: Proteins were obtained from the organic phase of thepreviously described TRI Reagent mRNA extraction. After ethanoladdition, samples were centrifuged at 4.600 rpm for 5 min at 4° C. Thesupernatant was transferred to a new tube, incubated with isopropanolfor 10 min at RT, and centrifuged at 12,000 g for 10 min at 4° C. Afterwashing the pellet three times with guanidine hydrochloride in 95%ethanol, it was disaggregated with a manual homogenizer for 30 sec andincubated for 10 min at RT. Every sample was centrifuged for 5 min at8,000 g at 4° C., the pellet disaggregated in 100% ethanol, incubated 20min at RT and centrifuged again for 5 min at 8,000 g at 4° C. Dry pelletwas solubilized in 4M Urea in 1% SDS at 40° C. for 15-20 min andcentrifuged for 10 min at 10,400 g at 4° C. The total proteinconcentration of each sample was determined by Bio-Rad DC™ ProteinAssay. Protein lysates were mixed with Laemmli sample buffer (5×) for afinal protein concentration of 1 μg/ml, heated for 5 min at 100° C. andcentrifuged for 5 min at 3,000 rpm at 4° C.

Gel electrophoresis was carried out in 12.5% sodium dodecyl sulfate(SDS)-polyacrylamide gels and resolved proteins were transferred to PVDFmembranes, which were blocked with 5% skimmed milk to avoid nonspecificbinding. Membranes were incubated in the following primary antibodies:goat anti-PTN (1:300) from R&D systems, Inc. (AF-252-PB) and mouseanti-GAPDH (1:2,000) from Santa Cruz Biotechnology, Inc. Heidelberg,Germany (SC-32233). After extensive washings in TBS-T (TBS/0.05% Tween20), membranes were incubated with the following horseradish peroxidaseconjugated secondary antibodies: rabbit anti-goat IgG (1:50,000) andgoat anti-mouse IgG (1:50,000), both from Sigma-Aldrich Co., Missouri,USA. Secondary antibodies were detected with ECL Advance kit (GEHealthcare Europe GmbH, Munich, Germany). Blot quantifications wereperformed by densitometric scanning using Scion Image Software. Thedensitometry values were normalized with respect to the values obtainedwith anti-GAPDH antibody. Data are expressed as mean±standard error.

An increase in PTN expression levels in the hippocampus (181±25, n=6,p<0.05) and cerebral cortex (190±16, n=6, p<0.05) of transgenic micewere shown (FIG. 3e ).

b) Immunohistochemistry: Male mice were anesthetized with 0.2 mlpentobarbital (50 mg/kg) and transcardially perfused (1: 75 ml NaCl0.9%; 2: 75 ml paraformaldehyde 4%). After brain extraction, 4 hourpost-fixation was carried out with paraformaldehyde 4% and the brainswere incubated in sacarose 30% for 36 hours. Once dry and frozen, 40 μmcoronal sections were cut along the hippocampus and the cerebral cortex.Immunohistochemistry was performed as following standard protocols andthe following antibodies were used: goat anti-PTN primary antibody(1:200) from R&D Systems (Ref. AF-252-PB) and donkey anti-goat secondarybiotinylated antibody (1:200) from Jackson Immunoresearch (Ref.705-065-147). PTN labeled regions give rise to a DAB precipitate whenanalyzed under Carl Zeiss Axioskop 2 Plus optic microscope.

Transgenic animals show a higher intensity signal of PTN expression,compared to their wild-type littermates, both in the hippocampus and thecerebral cortex (FIG. 5).

Example 6 Behavioural Testing

It is possible to detect whether or not a transgenic mouse shows anxiousand/or depressive symptoms by conventional methods of measuring anxietyand/or depression-related disorders, such as the following methodsdescribed in items 1) to 4):

1) General Observation: Irwin Test Adaptation

A general observation of animal behavior was carried out, such asmeasurements of weight, rectal temperature, excitation, fear and jumpingresponse to touch, vocalization to touch, aggressiveness, tremor, motortics, respiratory rate and ptosis. These observations were performed 5min after placing the animals in a transparent box (20×20×30 cm) with 50lux light illumination.

No significant differences were detected in animal's weight and rectaltemperature between wild-type and transgenic mice. FIG. 6 summarizes theresults obtained for the rest of the characteristics studied.

These results suggest that PTN-OE mice are more prone to be reactive toexternal stimuli, and they show a higher level of excitation thanwild-type mice.

2) Motor activity-open field test: The objective of this experiment wasto evaluate the possible influence of the locomotor responses in thebehavioral phenotype of transgenic mice overexpressing PTN. For thispurpose, locomotor activity was evaluated in wild-type and PTN-OE miceunder non-stressful conditions using locomotor activity boxes (FIG. 7a-c) and under stressful conditions using a high-illuminated open field(FIG. 7d-i ).

—Locomotor Activity Boxes:

Spontaneous horizontal and vertical locomotor activities were measuredduring three consecutive days under non-stressful conditions by usingindividual small Plexiglas activity boxes (9×20×11 cm, Imetronic,France). Each box contained a line of photocells 2 cm above the floor tomeasure horizontal locomotor activity, and another line located 6 cmabove the floor to measure vertical activity (rearing). Mice were placedin the boxes and total activity, horizontal activity and number ofrearings were recorded during 30 min in a low luminosity environment(20±25 lux). Data obtained in the locomotor activity boxes were analyzedby using a two-way analysis of variance (ANOVA) with genotype (wild-typeor transgenic) as between-subjects factor and day as within-subjectfactor. Differences were considered significant at P<0.05. All resultsare expressed as mean±S.E.M. The statistical analysis was performedusing SPSS software version 15.0 (SPSS Inc., Chicago, Ill., USA).

Total activity, horizontal activity and number of rearings wereevaluated in wild-type and transgenic mice overexpressing PTN duringthree consecutive days in the locomotor activity boxes. Two-way ANOVArevealed a significant effect of day [F_((2,74))=71.68, P<0.001] ontotal activity, whereas non-significant effects of genotype[F_((1,37))=0.09, n.s] nor interaction between day and genotype[F_((2,74))=2.32, n.s.] were obtained (FIG. 7a ). Similarly, two-wayANOVA revealed a significant effect of day [F_((2,74))=66.41, P<0.001]on horizontal locomotor activity, without effect of genotype[F_((1,37))=0.03, n.s], nor interaction between both factors[F_((2,74))=0.40, n.s.] (FIG. 7b ). In agreement, two-way ANOVA revealeda significant effect of day [F_((2,74))=50.96, P<0.001] on verticalactivity, without significant effect of genotype [F_((1,37))=0.55, n.s],nor interaction between both factors [F_((2,74))=0.78, n.s.] (FIG. 7c ).These results indicate that both wild-type and transgenic mice similarlydecreased locomotor activity across days, as a result of the expectedhabituation to the new environment. Interestingly, wild-type andtransgenic mice showed similar responses on all the locomotor parametersevaluated under these non-stressful conditions suggesting that the basallocomotor activity is similar in both genotypes.

—Open Field:

Locomotor activity was also evaluated under stressful conditions byusing an open field. The open field was a white rectangular area (70 cmwide, 90 cm long and 60 cm high) brightly illuminated from the top (500lux). A total of 63 squares (10×10 cm) were drawn with black lines onthe white floor of the field. Six events were recorded during anobservation period of 5 min: time spent in central area, crossingsquares in central area, entries in central area, crossing squares inperipheral area, number of rearings and number of groomings. Dataobtained in the open field were analyzed by using a one-way ANOVA withgenotype (wild-type or transgenic) as between-subjects factor.Differences were considered significant at P<0.05. All results areexpressed as mean±S.E.M. The statistical analysis was performed usingSPSS software version 15.0 (SPSS Inc., Chicago, Ill., USA).

The following behavioural responses were evaluated in the open field:time spent in central area, crossing squares in central area, entries incentral area, crossing squares in peripheral area, number of rearingsand number of groomings. One-way ANOVA revealed a significant effect ofgenotype on the time spent in the central area [F_((1,37))=8.23,P<0.01], crossing squares in the central area [F_((1,37))=6.31, P<0.05]and number of groomings [F_((1,37))=6.24, P<0.05]. Indeed, transgenicmice spent lower time in the central area, lower number of crossingsquares in the central area and higher number of groomings thanwild-type mice (FIGS. 7d, e and h ). An enhanced grooming behaviour anda decreased exploration of the central area under these stressfulconditions would reflect an enhanced anxiety-like behaviour in thesetransgenic mice. In agreement with the results obtained in the locomotoractivity boxes, one-way ANOVA did not show significant differencesbetween genotypes on crossing squares in the peripheral area[F_((1,37))=1.95, n.s] and total number of rearings [F_((1,37))=0.07,n.s] (FIGS. 4g and i ). These two parameters mainly reflect basallocomotion and exploration under these experimental conditions,suggesting that locomotor responses are not modified in the transgenicmice. Furthermore, one-way ANOVA did not reveal significant differencesbetween genotypes on the number of entries in the central area[F_((1,37))=0.01, n.s] (FIG. 4f ). Taken together, the results in thisbehavioural paradigm suggest that basal locomotor responses are notmodified in transgenic mice under stressful conditions, whereas thesemice exhibited an anxiety-like behavior.

3) Assessment of Anxiety-Like Behaviours

—Elevated Plus Maze

The elevated plus-maze consists of a black Plexiglas apparatus with fourarms (16 cm long×5 cm wide) set in cross from a neutral central square(5×5 cm). Two opposite arms are delimited by vertical walls (closedarms), while the two other opposite arms have unprotected edges (openarms). The maze is elevated 40 cm above the ground and placed inindirect light (100 Lux) at 22° C. At the beginning of the 5 minobservation session, each mouse is placed in the central neutral zone,facing one of the open arms. The total numbers of visits to the closedand open arms, and the time spent in the closed and open arms, are thenobserved on a monitor through a video camera system (ViewPoint, France).The maze was cleaned between sessions using 70% ethanol.

In the elevated plus maze, transgenic mice spent less time and theywalked less distance in the open arm compared to their wild-typelittermates, (the two-tailed one-sample t-test, p<0.002 and p<0.05respectively) (FIGS. 8b and 8c ). Similarly, transgenic mice showed adecrease but not statistically significant, in the number of entrieswhen compared with wild-type (the two-tailed one-sample t-test, p=0.06)(FIG. 8a ). These data are indicative of an anxiety-like behaviour.

—Light-Dark Box

The box consists of a small (15×20×25 cm) dimly lit (5 lux) compartment,with black walls and a black floor, connected by a 4 cm long tunnelleading to a larger compartment (30×20×25 cm) intensely lit (500 lux)with white walls and white floor. Lines are drawn on the floor of bothcompartments to allow measurement of locomotor activity by counting thenumber of squares (5×5 cm) crossed. Floor lines separate the litcompartment into three equal zones, from the tunnel to the oppositewall, designated as proximal, median and distal zone. Each animal isplaced in the dark compartment facing the tunnel at the beginning of theobservation session. Latency to go for the first time to the litcompartment, time spent, number of squares crossed and number of visitsinto each zone of the lit compartment are recorded for 5 min through avideocamera system (ViewPoint, France).

PTN over-expressing transgenic animals avoided facing the litcompartment of the box (the two-tailed one-sample t-test, p<0.05) (FIG.8e ) and showed a decrease in the number of squared crossed (thetwo-tailed one-sample t-test, p=0.06) (FIG. 8f ). No changes were foundin the latency to go for the first time to the lit compartment (FIG. 8d). Considering as variables both, zone and genotype, significantdifferences were found between zone visits (two-way Anova,F(2,108)=17.93) and the interaction genotype-zone (two-way Anova,F(4,108)=2.533) (FIG. 8g ).

4) Assessment of Depressive-Like Behaviours

—Tail Suspension Test (TST)

Mice are individually suspended by adhesive tape, 1 cm from the tip ofthe tail 50 cm above a bench top for a 6 min period. The time that theanimal is totally inactive during this period is recorded.

Transgenic animals spent more time immobile than their wild-typelittermates (the two-tailed one-sample t-test, p<0.05) (FIG. 8h ,suggesting a depressive-like behavior.

—Sucrose Intake Test

Mice are habituated for 2 days to drink a sucrose solution (1% sucrosein water in graduated feeding bottles with an anti-dripping system). 24hours after drinking deprivation, the amount of sucrose intake isrecorded for 1 hour.

Transgenic mice tend to intake less amount of sucrose compared to theirwild-type littermates, although differences are not statisticallysignificant (the two-tailed one-sample t-test, p=0.1) (FIG. 8i ). Alower intake could indicate anhedonia, which is a feature related todepressive-like behaviors.

Example 7 Long Term Potentiation (LTP)

We wished to analyze the involvement of PTN in the induction andmaintenance of the LTP in hippocampal slices from transgenic andwild-type animals.

All the animals used for this study were killed by decapitation.Immediately after decapitation, animal brains were removed and droppedinto a bubbled (95% 02 and 5% CO2) and ice-cold Krebs-Ringer bicarbonate(KRB) solution containing, in mM; 109 NaCl, 2.5 KCl, 1 KH₂PO₄, 1.3MgSO₄, 2.5 CaCl₂, 26.2 NaHCO₃ and 11 glucose. As it has been previouslydescribed (del Olmo et al., 2003), transverse slices (400 μm) of eachhippocampus were cut with a manual tissue chopper and placed in ahumidified interface chamber at room temperature (20-25° C.). After 2-hincubation period, slices were transferred to the submersion recordingchamber in which it was continuously perfused with a standard KRBsolution at 1.8-2 ml/min rate. Field excitatory postsynaptic potentials(fEPSPs) were recorded for 80 min in CA1 stratum radiatum with tungstenelectrodes (1 MC) and evoked by stimulating Schaffercollateral-commisural fibers with biphasic electrical pulses (30-70 μA;100 μs; 0.033 or 0.066 Hz) delivered through bipolar tungsten insulatedmicroelectrodes (0.5 MΩ). The recording electrode was connected to anAl-402 amplifier (Axon Instruments, USA) plugged to a CyberAmp 320signal conditioner (Axon Instruments, USA). Electrical pulses weresupplied by a pulse generator Master 8 (AMPI, Israel). Evoked responseswere digitized at 25-50 Hz using a Digidata 1320A (Axon Instruments,USA) and stored on a Pentium IV IBM compatible computer using pCLAMP 9.0software (Axon Instruments, USA).

The Schaffer collateral groups of axons contacting over the populationof cells were stimulated every 15 s. After obtaining stable synapticresponses for at least 20 min (baseline period), CA1 area was tetanizedwith three 100 Hz pulses for 1 s and 100 μs of duration every 20 sec(HFS). The synaptic strength was assessed by measuring the initial slopeof the fEPSP that was analyzed by means of pCLAMP 9.0 software. Datawere normalized with respect to the mean values of the responsesobtained by each animal at the 20 min baseline period. A single slicefrom each separate animal was considered as n=1. Allelectrophysiological experiments were carried out at 31-32° C.

The averaged values of the initial slope of the fEPSP were analyzed by arepeated measure ANOVA. Unpaired t-tests were performed for assessingspecific group differences in the average of the fEPSP measured during 5consecutive minutes at different times of the recording assay. In allthe cases, statistical differences were considered significant if theprobability of error was less than 5%. All calculations were performedusing SPSS statistical package 15.0 version.

PTN over-expressing mice presented important deficits in LTPestablishment. After HFS application, fEPSP slope increase was identicalin both groups (no statistical differences in the potentiation in the 5minutes alter HFS application). Nevertheless, transgenic PTN mice didnot maintain the potentiation until the end of the recording. Theseresults indicate that this mice line presented the impairment of LTPmaintenance respect to control (F[1,58]=16,67; p<0,001 from HFSapplication until the final of the recording) (FIG. 9).

These results may suggest deficits in processes involved in themaintenance of the phenomenon, such us protein synthesis. This could endup in long term memory problems.

Example 8 Method of Screening Agents as Potential Anxiety and/orDepression Therapeutics

A potential therapeutic agent for the treatment of anxiety and/ordepression can be tested by administering the test substance to atransgenic animal of the invention. Following the administration of thesubstance, the behavioural tests (for example behavioural tests asdisclosed in Example 6 above) are repeated, in order to evaluate whetherthe compound modifies the results observed prior to administration ofthe compound. Preferably, the one or more behavioural tests areadditionally performed on one or more untreated transgenic animals ofthe invention to act as controls.

A potential therapeutic agent may be classified as a candidate forfurther investigation on the basis of a positive screening result. Apositive screening result may be one in which the test agent is found torestore or normalise a behavioural outcome of an animal of the inventionto or towards the behavioural outcome of a normal animal (e.g. an animalnot having altered levels of PTN but otherwise identical to the animalof the invention). In some cases, the behavioural outcome may bequantified and the degree of restoration or normalisation of thebehavioural outcome measured. In this way a pre-determined thresholdlevel may be set to allow classification of an agent being screened aspositive or negative for restoration or normalisation of behaviour.Agents classified as positive in this way may then be subjected tofurther animal testing and/or human clinical testing for safety and/orefficacy in the treatment of a psychiatric illness, e.g. anxiety and/ordepression.

The substances which can be used for the screening method are notlimited, but may include for example: commercially available compounds(GSK-1838705A (Sabbatini et al., 2009); Crizotinib (Christensen et al.,2007); Imatinib (Heinrich et al., 2003)), various known compoundsregistered in compound databases (Huang et al., 2003), compoundsobtained by combinatorial chemical files, compounds obtained bycombinatorial chemistry techniques, or chemically or biologicallymodified compounds derived from other compounds, culture supernatants ofmicroorganisms, natural components derived from plants or marineorganisms, animal tissue, interference RNA (Calvet et al., 2006; Yao etal., 2011), peptides (Hamma-Kourbali et al., 2008; Diamantopoulou etal., 2010; Mikelis et al., 2011), splice forms (Lorente et al., 2005)and antibodies (Chen et al., 2007). Preferably, the agent is a compoundcapable of passing through the blood-brain barrier (BBB) such that it is“centrally active”.

Preferably, one or more positive control compounds, which are known tohave therapeutic activity in the treatment of psychiatric illness,particularly depression, are used in the screening method as a referenceagainst which to compare the effects of any test agent. Preferredpositive control compounds include Tricyclic Antidepressant such asImipramine and Amitriptiline; Selective Serotonin Reuptake inhibitors(SSRI) such as Fluoxetine, Paroxetine, Sertraline, Citalopram,Dapoxetine, Escitalopram, Fluvoxamine and Vilazodone; Serotoninnoradrenaline reuptake inhibitors (SNRI) such as Duloxetine andVelafaxine.

Example 9 Novelty Suppressed Feeding Test Assesses Anxiety and/orDepression

Novelty suppressed feeding test assesses anxiety and/or depression bymeasuring the latency of an animal to approach and eat food in a novelenvironment following chronic treatment with antidepressants. Mice(WT=14 and TG=14) were separated in two experimental groups: Withoutfluoxetine treatment and with fluoxetine treatment. The Fluoxetine wasdelivered ad libitum in the drinking water at a concentration of 160mg/I (approximately doses of 20-25 mg/kg/day) during 4 weeks. Mice wereweighed and food was removed from their cage. Twenty four hr afterremoval of food, mice were transferred to the testing room, placed in aclean holding cage and allowed to habituate for at least 30 min. Thetesting apparatus consisted of a clear Plexiglas open-field (50×50×25cm) under an aversive condition (100 lux luminosity). The floor wascovered with 2 cm of corncob bedding. A small piece of mouse chow wasplaced in the center of the arena. At the start of the experiment, eachmouse was placed in the corner of the testing area, and the time to thefirst feeding event was recorded. After all mice from a single cage weretested, mice were returned to their home cage. A longer latency to feedreflects an anxiety-like behavior.

Evaluation of homogeneity of variance (Levene's test) showed that thisassumption is violated, thus a rank transformation was applied on data.Measurement of Latency to feed were rank-transformed (Conover and Iman,1981) and subjected to a Two-way ANOVA having two levels of genotype(wild-type or WT and transgenic or TG) and two levels of treatment(without fluoxetine or -Flx and with fluoxetine or +Flx). The maineffect of genotype was not significant (F_((1,24))=0.001, n.s.).However, the main effect of treatment was significant(F_((1,24))=21.080, P=0.00012). In addition to this, there is asignificant interaction between the two factors (F_((1,24))=7.624,P=0.011).

In base to results that show a positive interaction between treatmentand genotype, rank-transformed data were submitted to One-way ANOVA tostudy the effect of treatment on genotype. No significant differences(F_((1,24))=1.675, n.s.) were found in WT when comparing treated versusuntreated samples, while a significant difference was found in TG mice(F_((1,24))=27.029, P<0.00003). The differences between genotypes in thetreated (F_((1,24))=3.901, P=0.06) or the untreated group((F_((1,24))=3.724, P=0.07) were not significant, but both comparisonshowed a trend to increase the latency to feed when compare with WT.

The results showed that PTN overexpression in mouse brain induceanxiety-like responses that are reversed after a chronic treatment withFluoxetine. Furthermore, Tg mice were more sensitive to the effect ofFluoxetine when compared with Wt.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety.

The specific embodiments described herein are offered by way of example,not by way of limitation. Any sub-titles herein are included forconvenience only, and are not to be construed as limiting the disclosurein any way.

TABLE 1 Final list of 383 selected genes Affymetrix ID Gene Title GeneSymbol FC DEP 208047_s_at NGFI-A binding protein 1 (EGR1 bindingprotein 1) NAB1 0.37 217663_at zinc finger protein 234 ZNF234 0.44220122_at multiple C2-domains with two transmembrane regions 1 MCTP10.46 218326_s_at leucine-rich repeat-containing G protein-coupledreceptor 4 LGR4 0.49 202203_s_at autocrine motility factor receptor AMFR0.50 202478_at tribbles homolog 2 (Drosophila) TRIB2 0.52 202666_s_atactin-like 6A ACTL6A 0.54 201816_s_at glioblastoma amplified sequenceGBAS 0.54 206652_at zinc finger protein 237 ZNF237 0.54 201424_s_atcullin 4A CUL4A 0.57 213067_at myosin, heavy polypeptide 10, non-muscleMYH10 0.57 201601_x_at interferon induced transmembrane protein 1 (9-27)IFITM1 0.60 209298_s_at intersectin 1 (SH3 domain protein) ITSN1 0.60221920_s_at mitochondrial solute carrier protein MSCP 0.60 210383_atsodium channel, voltage-gated, type I, alpha subunit SCN1A 0.60215772_x_at succinate-CoA ligase, GDP-forming, beta subunit SUCLG2 0.60209116_x_at hemoglobin, beta /// hemoglobin, beta HBB 0.61 209684_at Rasand Rab interactor 2 RIN2 0.61 207996_s_at chromosome 18 open readingframe 1 C18orf1 0.62 217232_x_at hemoglobin, beta HBB 0.62 221760_atMannosidase, alpha, class 1A, member 1 MAN1A1 0.62 206935_atprotocadherin 8 PCDH8 0.62 216769_x_at Chromosome 9 open reading frame150 C9orf150 0.63 213212_x_at golgin A6 family-like 4 GOLGA6L4 0.63211696_x_at hemoglobin, beta /// hemoglobin, beta HBB 0.63 222240_s_atmyo-inositol 1-phosphate synthase A1 ISYNA1 0.63 215791_at Intersectin 1(SH3 domain protein) ITSN1 0.63 209034_at proline-rich nuclear receptorcoactivator 1 PNRC1 0.63 202964_s_at regulatory factor X, 5 (influencesHLA class II expression) RFX5 0.63 209648_x_at suppressor of cytokinesignaling 5 SOCS5 0.63 87100_at Abhydrolase domain containing 2 ABHD20.64 210066_s_at aquaporin 4 AQP4 0.64 213018_at GATA zinc finger domaincontaining 1 GATAD1 0.64 217414_x_at hemoglobin, alpha 2 HBA2 0.64209316_s_at HBS1-like (S. cerevisiae) HBS1L 0.64 211990_at majorhistocompatibility complex, class II, DP alpha 1 HLA-DPA1 0.64 218578_athyperparathyroidism 2 (with jaw tumor) HRPT2 0.64 219564_at potassiuminwardly-rectifying channel, subfamily J, member 16 KCNJ16 0.64212942_s_at KIAA1199 KIAA1199 0.64 214658_at transmembrane emp24 proteintransport domain containing 7 TMED7 0.64 221645_s_at zinc finger protein83 (HPF1) ZNF83 0.64 204273_at endothelin receptor type B EDNRB 0.65218053_at formin binding protein 3 FNBP3 0.65 203222_s_attransducin-like enhancer of split 1 (E(sp1) homolog, Drosophila) TLE10.65 204520_x_at bromodomain containing 1 BRD1 0.66 201842_s_atEGF-containing fibulin-like extracellular matrix protein 1 EFEMP1 0.66209341_s_at inhibitor of kappa light polypeptide gene enhancer inB-cells, kinase IKBKB 0.66 beta 201243_s_at ATPase, Na+/K+ transporting,beta 1 polypeptide ATP1B1 0.67 201617_x_at caldesmon 1 CALD1 0.67202653_s_at membrane-associated ring finger (C3HC4) 7 MARCH7 0.67208993_s_at peptidyl-prolyl isomerase G (cyclophilin G) PPIG 0.67211074_at Folate receptor 1 (adult) Hs.73769 0.68 214071_atMetallophosphoesterase 1 MPPE1 0.68 206552_s_at tachykinin, precursor 1(substance K, substance P, neurokinin 1, TAC1 0.68 neurokinin 2,neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma)209386_at transmembrane 4 L six family member 1 TM4SF1 0.68 222316_atVesicle docking protein p115 VDP 0.68 219376_at zinc finger protein 322BZNF322B 0.68 210067_at aquaporin 4 AQP4 0.69 202425_x_at proteinphosphatase 3 (formerly 2B), catalytic subunit, alpha isoform PPP3CA0.69 (calcineurin A alpha) 209466_x_at pleiotrophin (heparin bindinggrowth factor 8, neurite growth- PTN 0.69 promoting factor 1)205651_x_at Rap guanine nucleotide exchange factor (GEF) 4 RAPGEF4 0.69212451_at SECIS binding protein 2-like SECISBP2L 0.69 201123_s_ateukaryotic translation initiation factor 5A EIF5A 0.70 219445_at gliomatumor suppressor candidate region gene 1 GLTSCR1 0.70 218604_at LEMdomain containing 3 LEMD3 0.70 218049_s_at mitochondrial ribosomalprotein L13 MRPL13 0.70 213725_x_at xylosyltransferase I XYLT1 0.71221008_s_at alanine-glyoxylate aminotransferase 2-like 1 ///alanine-glyoxylate AGXT2L1 0.72 aminotransferase 2-like 1 212224_ataldehyde dehydrogenase 1 family, member A1 ALDH1A1 0.72 214553_s_atcyclic AMP phosphoprotein, 19 kD ARPP-19 0.72 206253_at discs, largehomolog 2, chapsyn-110 (Drosophila) DLG2 0.72 201667_at gap junctionprotein, alpha 1, 43 kDa (connexin 43) GJA1 0.72 211596_s_atleucine-rich repeats and immunoglobulin-like domains 1 /// leucine-LRIG1 0.72 rich repeats and immunoglobulin-like domains 1 219126_at PHDfinger protein 10 PHF10 0.72 212526_at spastic paraplegia 20, spartin(Troyer syndrome) SPG20 0.72 202796_at synaptopodin SYNPO 0.72 212976_atT-cell activation leucine repeat-rich protein TA-LRRP 0.72 203527_s_atadenomatosis polyposis coli APC 0.73 215460_x_at bromodomain containing1 BRD1 0.73 214198_s_at DiGeorge syndrome critical region gene 2 DGCR20.73 214022_s_at interferon induced transmembrane protein 1 (9-27)IFITM1 0.73 203354_s_at pleckstrin and Sec7 domain containing 3 PSD30.73 200971_s_at stress-associated endoplasmic reticulum protein 1 SERP10.73 203583_at unc-50 homolog (C. elegans) UNC50 0.73 217975_at WWdomain binding protein 5 WBP5 0.73 207606_s_at Rho GTPase activatingprotein 12 ARHGAP12 0.74 203502_at 2,3-bisphosphoglycerate mutase ///2,3-bisphosphoglycerate mutase BPGM 0.74 214214_s_at complementcomponent 1, q subcomponent binding protein C1QBP 0.74 218930_s_athypothetical protein FLJ11273 FLJ11273 0.74 214414_x_at hemoglobin,alpha 2 /// hemoglobin, alpha 2 HBA2 0.74 208965_s_at interferon,gamma-inducible protein 16 IFI16 0.74 32069_at Nedd4 binding protein 1N4BP1 0.74 203478_at NADH dehydrogenase (ubiquinone) 1, subcomplexunknown, 1, 6 kDa NDUFC1 0.74 208638_at protein disulfideisomerase-associated 6 PDIA6 0.74 209025_s_at synaptotagmin binding,cytoplasmic RNA interacting protein SYNCRIP 0.74 211699_x_at hemoglobin,alpha 1 /// hemoglobin, alpha 1 /// hemoglobin, alpha 2 HBA1 0.75 ///hemoglobin, alpha 2 207480_s_at Meis1, myeloid ecotropic viralintegration site 1 homolog 2 (mouse) MEIS2 0.75 204491_atPhosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce PDE4D0.75 homolog, Drosophila) 219196_at secretogranin III SCG3 0.75202272_s_at F-box protein 28 FBXO28 0.76 204018_x_at hemoglobin, alpha 1/// hemoglobin, alpha 1 /// hemoglobin, alpha 2 HBA1 0.76 ///hemoglobin, alpha 2 218352_at regulator of chromosome condensation(RCC1) and BTB (POZ) RCBTB1 0.76 domain containing protein 1 215452_x_atSMT3 suppressor of mif two 3 homolog 4 (yeast) SUMO4 0.76 205139_s_aturonyl-2-sulfotransferase UST 0.76 202850_at ATP-binding cassette,sub-family D (ALD), member 3 ABCD3 0.77 203540_at glial fibrillaryacidic protein GFAP 0.77 218558_s_at mitochondrial ribosomal protein L39MRPL39 0.77 208731_at RAB2, member RAS oncogene family RAB2 0.77221493_at TSPY-like 1 TSPYL1 0.77 212074_at unc-84 homolog A (C.elegans) UNC84A 0.77 212200_at nkyrin repeat and LEM domain containing 2ANKLE2 0.78 202221_s_at E1A binding protein p300 EP300 0.78 209291_atinhibitor of DNA binding 4, dominant negative helix-loop-helix proteinID4 0.78 34031_i_at KRIT1, ankyrin repeat containing KRIT1 0.78207121_s_at mitogen-activated protein kinase 6 MAPK6 0.78 203910_atPTPL1-associated RhoGAP 1 PARG1 0.78 205361_s_at prefoldin 4 PFDN4 0.78214435_x_at v-ral simian leukemia viral oncogene homolog A (ras related)RALA 0.78 202277_at serine palmitoyltransferase, long chain base subunit1 SPTLC1 0.78 200887_s_at signal transducer and activator oftranscription 1, 91 kDa STAT1 0.78 202573_at casein kinase 1, gamma 2CSNK1G2 0.79 201921_at guanine nucleotide binding protein (G protein),gamma 10 GNG10 0.79 218239_s_at GTP binding protein 4 GTPBP4 0.79209458_x_at hemoglobin, alpha 1 /// hemoglobin, alpha 1 /// hemoglobin,alpha 2 HBA1 0.79 /// hemoglobin, alpha 2 205051_s_at v-kitHardy-Zuckerman 4 feline sarcoma viral oncogene homolog KIT 0.79208102_s_at pleckstrin and Sec7 domain containing PSD 0.79 211733_x_atsterol carrier protein 2 /// sterol carrier protein 2 SCP2 0.79208389_s_at solute carrier family 1 (glial high affinity glutamatetransporter), SLC1A2 0.79 member 2 202690_s_at small nuclearribonucleoprotein D1 polypeptide 16 kDa SNRPD1 0.79 201448_at TIA1cytotoxic granule-associated RNA binding protein TIA1 0.79 218617_attRNA isopentenyltransferase 1 TRIT1 0.79 218096_at1-acylglycerol-3-phosphate O-acyltransferase 5 (lysophosphatidic AGPAT50.80 acid acyltransferase, epsilon) 221596_s_at chromosome 7 openreading frame 64 C7orf64 0.80 221804_s_at family with sequencesimilarity 45, member B /// family with sequence FAM45B 0.80 similarity45, member A 214429_at myotubularin related protein 6 MTMR6 0.80217764_s_at RAB31, member RAS oncogene family RAB31 0.80 212042_x_atribosomal protein L7 RPL7 0.80 202800_at solute carrier family 1 (glialhigh affinity glutamate transporter), SLC1A3 0.80 member 3 212725_s_athypothetical protein TI-227H TI-227H 0.80 208374_s_at capping protein(actin filament) muscle Z-line, alpha 1 CAPZA1 0.81 202469_s_at cleavageand polyadenylation specific factor 6, 68 kDa CPSF6 0.81 209630_s_atF-box and WD-40 domain protein 2 FBXW2 0.81 212334_at glucosamine(N-acetyl)-6-sulfatase (Sanfilippo disease IIID) GNS 0.81 218641_atchromosome 11 open reading frame 95 C11orf95 0.81 212120_at Ras homologgene family, member Q RHOQ 0.81 216274_s_at SEC11-like 1 (S. cerevisiae)SEC11L1 0.81 218878_s_at sirtuin (silent mating type informationregulation 2 homolog) 1 (S. cerevisiae) SIRT1 0.81 212774_at zinc fingerprotein 238 ZNF238 0.81 201334_s_at Rho guanine nucleotide exchangefactor (GEF) 12 ARHGEF12 0.82 201117_s_at carboxypeptidase E CPE 0.82218443_s_at DAZ associated protein 1 DAZAP1 0.82 207768_at early growthresponse 4 EGR4 0.82 217828_at modulator of estrogen inducedtranscription FLJ13213 0.82 221255_s_at transmembrane protein 93 TMEM930.82 201468_s_at NAD(P)H dehydrogenase, quinone 1 NQO1 0.82 221795_atneurotrophic tyrosine kinase, receptor, type 2 NTRK2 0.82 208205_atprotocadherin alpha 9 PCDHA9 0.82 203966_s_at protein phosphatase 1A(formerly 2C), magnesium-dependent, alpha PPM1A 0.82 isoform /// proteinphosphatase 1A (formerly 2C), magnesium- dependent, alpha isoform47069_at proline rich protein 5 PRR5 0.82 212468_at sperm associatedantigen 9 SPAG9 0.82 218466_at TBC1 domain family, member 17 TBC1D170.82 218090_s_at WD repeat domain 11 WDR11 0.82 218490_s_at zinc fingerprotein 302 ZNF302 0.82 209028_s_at abI-interactor 1 ABI1 0.83210068_s_at aquaporin 4 AQP4 0.83 221482_s_at cyclic AMP phosphoprotein,19 kD ARPP-19 0.83 53720_at chromosome 19 open reading frame 66 C19orf660.83 211745_x_at hemoglobin, alpha 1 /// hemoglobin, alpha 1 ///hemoglobin, alpha 2 HBA1 0.83 /// hemoglobin, alpha 2 200989_athypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix HIF1A0.83 transcription factor) 221847_at Transcribed locus, strongly similarto XP_001721967.1 PREDICTED: Hs.521817 0.83 similar to mCG115122 [Homosapiens] 221045_s_at period homolog 3 (Drosophila) PER3 0.83 202318_s_atSUMO1/sentrin specific protease 6 SENP6 0.83 213881_x_at SMT3 suppressorof mif two 3 homolog 2 (yeast) SUMO2 0.83 220990_s_at likely ortholog ofrat vacuole membrane protein 1 /// likely ortholog of VMP1 0.83 ratvacuole membrane protein 1 203608_at aldehyde dehydrogenase 5 family,member A1 (succinate- ALDH5A1 0.84 semialdehyde dehydrogenase)211502_s_at PFTAIRE protein kinase 1 PFTK1 0.84 219485_s_at proteasome(prosome, macropain) 26S subunit, non-ATPase, 10 PSMD10 0.84 212458_atsprouty-related, EVH1 domain containing 2 SPRED2 0.84 217853_attensin-like SH2 domain containing 1 TENS1 0.84 204847_at zinc finger andBTB domain containing 11 ZBTB11 0.84 213220_at BBSome interactingprotein 1 BBIP1 0.85 213294_at eukaryotic translation initiation factor2-alpha kinase 2 EIF2AK2 0.85 220188_at junctophilin 3 JPH3 0.85222158_s_at CGI-146 protein PNAS-4 0.85 200845_s_at peroxiredoxin 6PRDX6 0.85 200608_s_at RAD21 homolog (S. pombe) RAD21 0.85 212749_s_atring finger and CHY zinc finger domain containing 1 RCHY1 0.85 203908_atsolute carrier family 4, sodium bicarbonate cotransporter, member 4SLC4A4 0.85 201273_s_at signal recognition particle 9 kDa SRP9 0.85212388_at ubiquitin specific protease 24 USP24 0.85 203420_at familywith sequence similarity 8, member A1 FAM8A1 0.86 210111_s_at kelchdomain containing 10 KLHDC10 0.86 219156_at synaptojanin 2 bindingprotein SYNJ2BP 0.86 219935_at a disintegrin-like and metalloprotease(reprolysin type) with ADAMTS5 0.87 thrombospondin type 1 motif, 5(aggrecanase-2) 202834_at angiotensinogen (serine (or cysteine)proteinase inhibitor, clade A AGT 0.87 (alpha-1 antiproteinase,antitrypsin), member 8) 203295_s_at ATPase, Na+/K+ transporting, alpha 2(+) polypeptide ATP1A2 0.87 214334_x_at DAZ associated protein 2 ///similar to DAZ-associated protein 2 DAZAP2 0.87 (Deleted inazoospermia-associated protein 2) 217202_s_at glutamate-ammonia ligase(glutamine synthase) GLUL 0.87 200921_s_at B-cell translocation gene 1,anti-proliferative BTG1 0.88 217768_at chromosome 14 open reading frame166 C14orf166 0.88 211424_x_at methyltransferase like 7A METTL7A 0.88220924_s_at solute carrier family 38, member 2 SLC38A2 0.88 205152_atsolute carrier family 6 (neurotransmitter transporter, GABA), member 1SLC6A1 0.88 210512_s_at vascular endothelial growth factor VEGF 0.88214934_at ATPase, Class II, type 9B ATP9B 0.89 208853_s_at calnexin CANX0.89 209470_s_at glycoprotein M6A GPM6A 0.89 207700_s_at nuclearreceptor coactivator 3 NCOA3 0.89 209355_s_at phosphatidic acidphosphatase type 2B PPAP2B 0.89 212230_at phosphatidic acid phosphatasetype 2B PPAP2B 0.89 218284_at SMAD, mothers against DPP homolog 3(Drosophila) SMAD3 0.89 212625_at syntaxin 10 STX10 0.89 201857_at zincfinger RNA binding protein ZFR 0.89 219300_s_at contactin associatedprotein-like 2 CNTNAP2 0.90 206401_s_at microtubule-associated proteintau MAPT 0.90 218259_at MKL/myocardin-like 2 MKL2 0.90 203961_atnebulette NEBL 0.90 203177_x_at transcription factor A, mitochondrialTFAM 0.90 209656_s_at transmembrane protein 47 TMEM47 0.90 218185_s_atarmadillo repeat containing 1 ARMC1 0.91 206849_at gamma-aminobutyricacid (GABA) A receptor, gamma 2 GABRG2 0.91 217739_s_at pre-B-cellcolony enhancing factor 1 PBEF1 0.91 201594_s_at protein phosphatase 4,regulatory subunit 1 PPP4R1 0.91 202126_at PRP4 pre-mRNA processingfactor 4 homolog B (yeast) PRPF4B 0.91 220949_s_at chromosome 7 openreading frame 49 C7orf49 0.92 210015_s_at microtubule-associated protein2 MAP2 0.92 200969_at stress-associated endoplasmic reticulum protein 1SERP1 0.92 212190_at serine (or cysteine) proteinase inhibitor, clade E(nexin, plasminogen SERPINE2 0.92 activator inhibitor type 1), member 2213351_s_at transmembrane and coiled-coil domains 1 TMCC1 0.92 200972_attetraspanin 3 TSPAN3 0.93 201021_s_at destrin (actin depolymerizingfactor) DSTN 0.94 209956_s_at calcium/calmodulin-dependent proteinkinase (CaM kinase) II beta CAMK2B 0.95 207010_at gamma-aminobutyricacid (GABA) A receptor, beta 1 GABRB1 0.96 214953_s_at amyloid beta (A4)precursor protein (protease nexin-II, Alzheimer APP 0.97 disease)201445_at calponin 3, acidic CNN3 0.97 215145_s_at contactin associatedprotein-like 2 CNTNAP2 0.98 210906_x_at aquaporin 4 AQP4 0.99202438_x_at iduronate 2-sulfatase (Hunter syndrome) IDS 0.99 201427_s_atselenoprotein P, plasma, 1 SEPP1 0.99 210949_s_at eukaryotic translationinitiation factor 3, subunit 8, 110 kDa EIF3S8 1.00 221236_s_atstathmin-like 4 /// stathmin-like 4 STMN4 1.00 217717_s_at tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation YWHAB 1.00protein, beta polypeptide 213998_s_at DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 17 DDX17 1.01 201537_s_at dual specificity phosphatase 3(vaccinia virus phosphatase VH1- DUSP3 1.01 related) 200648_s_atglutamate-ammonia ligase (glutamine synthase) GLUL 1.01 205150_s_at TLR4interactor with leucine-rich repeats TRIL 1.01 200848_atS-adenosylhomocysteine hydrolase-like 1 AHCYL1 1.02 211547_s_atplatelet-activating factor acetylhydrolase, isoform Ib, alpha subunitPAFAH1B1 1.03 45 kDa 204230_s_at solute carrier family 17(sodium-dependent inorganic phosphate SLC17A7 1.03 cotransporter),member 7 201360_at cystatin C (amyloid angiopathy and cerebralhemorrhage) CST3 1.04 215363_x_at folate hydrolase (prostate-specificmembrane antigen) 1 FOLH1 1.04 203603_s_at zinc finger homeobox 1bZFHX1B 1.04 45572_s_at golgi associated, gamma adaptin ear containing,ARF binding protein 1 GGA1 1.05 217741_s_at zinc finger, A20 domaincontaining 2 ZA20D2 1.05 208840_s_at Ras-GTPase activating protein SH3domain-binding protein 2 G3BP2 1.06 200658_s_at prohibitin PHB 1.07208850_s_at Thy-1 cell surface antigen /// Thy-1 co-transcribed THY11.07 209372_x_at tubulin, beta 2 /// tubulin, beta polypeptide paralogTUBB2 1.07 207276_at cerebellar degeneration-related protein 1, 34 kDaCDR1 1.08 218532_s_at family with sequence similarity 134, member BFAM134B 1.08 208687_x_at heat shock 70 kDa protein 8 HSPA8 1.08201270_x_at NudC domain containing 3 NUDCD3 1.08 215773_x_at poly(ADP-ribose) polymerase family, member 2 PARP2 1.08 204218_at chromosome11 open reading frame 51 C11orf51 1.10 205549_at Purkinje cell protein 4PCP4 1.10 202004_x_at succinate dehydrogenase complex, subunit C,integral membrane SDHC 1.10 protein, 15 kDa 211714_x_at tubulin, betapolypeptide /// tubulin, beta polypeptide TUBB 1.10 207761_s_atmethyltransferase like 7A METTL7A 1.11 217871_s_at macrophage migrationinhibitory factor (glycosylation-inhibiting factor) MIF 1.11 210315_atsynapsin II SYN2 1.11 203545_at asparagine-linked glycosylation 8homolog (yeast, alpha-1,3- ALG8 1.12 glucosyltransferase) 209569_x_atDNA segment on chromosome 4 (unique) 234 expressed sequence D4S234E 1.1243511_s_at arrestin, beta 1 ARRB1 1.13 207722_s_at BTB (POZ) domaincontaining 2 BTBD2 1.13 200824_at glutathione S-transferase pi GSTP11.13 201627_s_at insulin induced gene 1 INSIG1 1.13 202138_x_at JTV1gene JTV1 1.13 218012_at TSPY-like 2 TSPYL2 1.13 213726_x_at tubulin,beta, 2 TUBB2 1.13 209517_s_at ash2 (absent, small, or homeotic)-like(Drosophila) ASH2L 1.14 217950_at nitric oxide synthase interactingprotein NOSIP 1.14 210131_x_at succinate dehydrogenase complex, subunitC, integral membrane SDHC 1.14 protein, 15 kDa 212238_at additional sexcombs like 1 (Drosophila) ASXL1 1.15 213052_at Protein kinase,cAMP-dependent, regulatory, type II, alpha PRKAR2A 1.15 200802_atseryl-tRNA synthetase SARS 1.15 203400_s_at transferrin TF 1.15208358_s_at UDP glycosyltransferase 8 (UDP-galactose ceramide UGT8 1.15galactosyltransferase) 214699_x_at WIPI49-like protein 2 WIPI-2 1.15200720_s_at ARP1 actin-related protein 1 homolog A, centractin alpha(yeast) ACTR1A 1.16 39835_at SET binding factor 1 SBF1 1.16 202118_s_atcopine III CPNE3 1.17 216850_at small nuclear ribonucleoproteinpolypeptide N SNRPN 1.17 217799_x_at ubiquitin-conjugating enzyme E2H(UBC8 homolog, yeast) UBE2H 1.17 205625_s_at calbindin 1, 28 kDa CALB11.18 200919_at polyhomeotic-like 2 (Drosophila) PHC2 1.18 212216_atprolyl endopeptidase-like PREPL 1.18 209875_s_at secreted phosphoprotein1 (osteopontin, bone sialoprotein I, early T- SPP1 1.18 lymphocyteactivation 1) 204260_at chromogranin B (secretogranin 1) CHGB 1.19218539_at F-box protein 34 FBXO34 1.19 202472_at mannose phosphateisomerase MPI 1.19 220653_at zinc finger, imprinted 2 ZIM2 1.19207593_at ATP-binding cassette, sub-family G (WHITE), member 4 ABCG41.20 218953_s_at prenylcysteine oxidase 1 like PCYOX1L 1.20 210130_s_attransmembrane 7 superfamily member 2 TM7SF2 1.20 222046_at arsenateresistance protein ARS2 ARS2 1.21 220725_x_at Dynein, axonemal, heavypolypeptide 3 DNAH3 1.21 204466_s_at synuclein, alpha (non A4 componentof amyloid precursor) /// SNCA 1.21 synuclein, alpha (non A4 componentof amyloid precursor) 207594_s_at synaptojanin 1 SYNJ1 1.21 207991_x_atacrosomal vesicle protein 1 ACRV1 1.22 218958_at chromosome 19 openreading frame 60 C19orf60 1.22 200647_x_at eukaryotic translationinitiation factor 3, subunit 8, 110 kDa EIF3S8 1.22 213629_x_atmetallothionein 1F (functional) MT1F 1.22 200003_s_at ribosomal proteinL28 /// ribosomal protein L28 RPL28 1.22 201870_at translocase of outermitochondrial membrane 34 TOMM34 1.22 202589_at thymidylate synthetaseTYMS 1.22 221676_s_at coronin, actin binding protein, 1C CORO1C 1.23209581_at HRAS-like suppressor 3 HRASLS3 1.23 209467_s_at MAP kinaseinteracting serine/threonine kinase 1 MKNK1 1.23 200820_at proteasome(prosome, macropain) 26S subunit, non-ATPase, 8 PSMD8 1.23 201127_s_atATP citrate lyase ACLY 1.24 202154_x_at tubulin, beta 3 TUBB3 1.24206966_s_at Kruppel-like factor 12 KLF12 1.25 217609_at B7 gene B7 1.26201953_at calcium and integrin binding 1 (calmyrin) CIB1 1.26217930_s_at toll interacting protein TOLLIP 1.26 211538_s_at heat shock70 kDa protein 2 HSPA2 1.28 219236_at progestin and adipoQ receptorfamily member VI PAQR6 1.28 205899_at cyclin A1 CCNA1 1.29 212878_s_atkinesin 2 60/70 kDa KNS2 1.29 220236_at pyruvate dehydrogenasephosphatase regulatory subunit PDPR 1.29 204035_at secretogranin II(chromogranin C) SCG2 1.29 210858_x_at ataxia telangiectasia mutated(includes complementation groups A, C ATM 1.30 and D) 207401_atprospero-related homeobox 1 PROX1 1.30 203022_at ribonuclease H2, largesubunit RNASEH2A 1.30 206748_s_at sperm associated antigen 9 SPAG9 1.30221196_x_at chromosome X open reading frame 53 CXorf53 1.31 218488_ateukaryotic translation initiation factor 2B, subunit 3 gamma, 58 kDaEIF2B3 1.31 209392_at ectonucleotide pyrophosphatase/phosphodiesterase 2(autotaxin) ENPP2 1.31 34868_at Est1p-like protein B EST1B 1.31207801_s_at ring finger protein 10 RNF10 1.31 212155_at ring fingerprotein 187 RNF187 1.31 206283_s_at T-cell acute lymphocytic leukemia 1TAL1 1.31 203842_s_at microtubule-associated protein, RP/EB family,member 3 MAPRE3 1.32 204179_at myoglobin MB 1.33 215771_x_at retproto-oncogene (multiple endocrine neoplasia and medullary RET 1.33thyroid carcinoma 1, Hirschsprung disease) 201785_at ribonuclease, RNaseA family, 1 (pancreatic) RNASE1 1.34 214915_at zinc finger protein 362ZNF362 1.34 212772_s_at ATP-binding cassette, sub-family A (ABC1),member 2 ABCA2 1.35 213300_at ATG2 autophagy related 2 homolog A (S.cerevisiae) ATG2A 1.35 212394_at KIAA0090 KIAA0090 1.35 210136_at Myelinbasic protein MBP 1.35 209283_at crystallin, alpha B CRYAB 1.36207972_at glycine receptor, alpha 1 (startle disease/hyperekplexia,stiff man GLRA1 1.36 syndrome) 217008_s_at glutamate receptor,metabotropic 7 GRM7 1.37 204550_x_at glutathione S-transferase M1 GSTM11.37 203436_at ribonuclease P/MRP 30 kDa subunit RPP30 1.37 201037_atphosphofructokinase, platelet PFKP 1.38 219105_x_at origin recognitioncomplex, subunit 6 homolog-like (yeast) ORC6L 1.39 222153_at myelinexpression factor 2 MYEF2 1.43 209771_x_at CD24 antigen (small cell lungcarcinoma cluster 4 antigen) CD24 1.45 222073_at collagen, type IV,alpha 3 (Goodpasture antigen) COL4A3 1.45 215643_at Sema domain,immunoglobulin domain (Ig), short basic domain, SEMA3D 1.45 secreted,(semaphorin) 3D 214063_s_at transferrin TF 1.45 218183_at chromosome 16open reading frame 5 C16orf5 1.46 210679_x_at — — 1.47 204073_s_atchromosome 11 open reading frame 9 C11orf9 1.48 204733_at kallikrein 6(neurosin, zyme) KLK6 1.50 206106_at mitogen-activated protein kinase 12MAPK12 1.51 204777_s_at mal, T-cell differentiation protein MAL 1.52208851_s_at Thy-1 cell surface antigen /// Thy-1 co-transcribed THY11.54 204719_at ATP-binding cassette, sub-family A (ABC1), member 8 ABCA81.55 266_s_at CD24 antigen (small cell lung carcinoma cluster 4 antigen)CD24 1.55 216617_s_at myelin associated glycoprotein MAG 1.55216379_x_at CD24 antigen (small cell lung carcinoma cluster 4 antigen)CD24 1.57 207713_s_at chromosome 20 open reading frame 18 C20orf18 1.58202068_s_at low density lipoprotein receptor (familialhypercholesterolemia) LDLR 1.60 215986_at Homo sapiens cDNA FLJ12058fis, clone HEMBB1002092. AK022120 1.61 205061_s_at exosome component 9EXOSC9 1.62 207445_s_at chemokine (C-C motif) receptor 9 CCR9 1.63212672_at Ataxia telangiectasia mutated (includes complementation groupsA, C ATM 1.66 and D) 215531_s_at gamma-aminobutyric acid (GABA) Areceptor, alpha 5 GABRA5 1.79 221031_s_at apolipoprotein L domaincontaining 1 APOLD1 2.01 220873_at RALBP1 associated Eps domaincontaining 2 REPS2 2.04

TABLE 2 Selected genes for qRT-PCR Gene Gene Name Symbol adrenergic,alpha-1A-, receptor ADRA1A adrenergic, alpha-1B-, receptor ADRA1Badrenergic, alpha-1D-, receptor ADRA1D adrenergic, alpha-2A-, receptorADRA2A amyloid beta (A4) precursor protein (protease nexin-II, APPAlzheimer disease) cyclic AMP phosphoprotein, 19 kD (Interim) ARPP-19axotrophin AXOT brain cell membrane protein 1 (Interim) BCMP1brain-derived neurotrophic factor BDNF chromosome 22 open reading frame4 C22orf4 calcium binding protein 1 (calbrain) CABP1calcium/calmodulin-dependent protein kinase (CaM kinase) CAMK2B II betacalnexin CANX chemokine (C-C motif) receptor 9 CCR9 cAMP-regulatedguanine nucleotide exchange factor II CGEF2 (Interim) chromogranin B(secretogranin 1) CHGB calcium and integrin binding 1 (calmyrin) CIB1CREB binding protein (Rubinstein-Taybi syndrome) CREBBP Gprotein-binding protein CRFG (Interim) CRFG corticotropin releasinghormone CRH corticotropin releasing hormone receptor 1 CRHR1corticotropin releasing hormone receptor 2 CRHR2 DNA segment onchromosome 4 (unique) 234 expressed D4S234E sequence dual-specificitytyrosine-(Y)-phosphorylation regulated DYRK2 kinase 2 ectonucleotidepyrophosphatase/phosphodiesterase 2 ENPP2 (autotaxin) erbb2 interactingprotein ERBB2IP gamma-aminobutyric acid (GABA) A receptor, alpha 1GABRA1 gamma-aminobutyric acid (GABA) A receptor, beta 1 GABRB1gamma-aminobutyric acid (GABA) A receptor, gamma 2 GABRG2 glialfibrillary acidic protein GFAP glycine receptor, alpha 1 (startledisease/hyperekplexia, GLRA1 stiff man syndrome) guanine nucleotidebinding protein 10 GNG10 glutamate receptor, metabotropic 7 GRM7 heatshock 70 kDa protein 2 HSPA2 5-hydroxytryptamine (serotonin) receptor 1AHTR1A 5-hydroxytryptamine (serotonin) receptor 1B HTR1B5-hydroxytryptamine (serotonin) receptor 1E HTR1E 5-hydroxytryptamine(serotonin) receptor 2A HTR2A 5-hydroxytryptamine (serotonin) receptor2C HTR2C integrin beta 3 binding protein (beta3-endonexin) ITGB3BPinositol 1,4,5-trisphosphate 3-kinase B ITPKB intersectin 1 (SH3 domainprotein) ITSN1 potassium voltage-gated channel, Shal-related subfamily,KCND2 member 2 potassium inwardly-rectifying channel, subfamily J,KCNJ16 member 16 putative L-type neutral amino acid transporter(Interim) KIAA0436 synaptopodin (Interim) KIAA1029 low densitylipoprotein receptor (familial LDLR hypercholesterolemia) myelinassociated glycoprotein MAG microtubule-associated protein 2 MAP2microtubule-associated protein, RP/EB family, member 3 MAPRE3microtubule-associated protein tau MAPT MAP kinase-interactingserine/threonine kinase 1 MKNK1 myelin-associated oligodendrocyte basicprotein MOBP NCK-associated protein 1 NCKAP1 neurotrophic tyrosinekinase, receptor, type 1 NTRK1 neurotrophic tyrosine kinase, receptor,type 3 NTRK3 opioid receptor, mu 1 OPRM1 polyhomeotic-like 2(Drosophila) PHC2 phosphatidic acid phosphatase type 2B PPAP2B proteinphosphatase 1A (formerly 2C), magnesium- PPM1A dependent, alpha isoformprotein phosphatase 2, regulatory subunit B (B56), beta PPP2R5B isoformprotein phosphatase 3 (formerly 2B), catalytic subunit, PPP3CA alphaisoform (calcineurin A alpha) protein phosphatase 4, regulatory subunit1 PPP4R1 protein kinase, cAMP-dependent, regulatory, type I, alphaPRKAR1A (tissue specific extinguisher 1) pleckstrin and Sec7 domainprotein PSD pleiotrophin (heparin binding growth factor 8, neurite PTNgrowth-promoting factor 1) protein tyrosine phosphatase, receptor type,M PTPRM RAB22A, member RAS oncogene family RAB22A regulator of G-proteinsignalling 4 RGS4 secretogranin II (chromogranin C) SCG2 sodium channel,voltage-gated, type I, alpha SCN1A solute carrier family 1 (glial highaffinity glutamate SLC1A2 transporter), member 2 solute carrier family 1(glial high affinity glutamate SLC1A3 transporter), member 3 solutecarrier family 38, member 2 SLC38A2 SM-11044 binding protein (Interim)SMBP somatostatin SST syntaxin 10 STX10 synapsin II SYN2 synaptojanin 1SYNJ1 synaptojanin 2 binding protein SYNJ2BP tachykinin, precursor 1(substance K, substance P, TAC1 neurokinin 1, neurokinin 2, neuromedinL, neurokinin alpha, neuropeptide K, neuropeptide gamma) TANK-bindingkinase 1 TBK1 transferrin TF

TABLE 3 Genes differentially expressed between cases and controls DEP vsControl Nombre p-value FC Amyloid beta (A4) precursor protein 0.00020.82 Cyclic AMP phosphoprotein, 19 kD 0.2461 0.76 Brain cell membraneprotein 1 0.0189 0.82 Chromosome 22 ORF 4 0.0103 0.88 Calcium bindingprotein 1 (calbrain) 0.0003 0.82 Calcium/calmodulin-dependent proteinkinase (CaM kinase) II beta 0.1101 0.92 Calnexin 0.0001 0.85cAMP-regulated guanine nucleotide exchange factor II 0.0055 0.80 Calciumand integrin biniding 1 (Calmyrin) 0.0000 0.76 G protein-binding protein0.0002 0.86 DNA segment on chromosome 4 (unique) 234 expressed sequence0.0411 1.12 (neuron-specific protein) Erbb2 interacting protein 0.15580.87 Guanine nucleotide binding protein 10 0.0127 0.65 Integrin beta 3binding protein 0.3006 0.86 Inositol 1,4,5-trisphosphate 3-kinase B0.0247 0.81 Intersectin 1 0.0125 0.91 Potassium voltage-gated channel,Shal-related subfamily, member 2 0.1109 0.92 Potassiuminwardly-rectifying channel, subfamily J, member 16 0.0248 0.74Synaptopodin 0.0104 0.85 Low density lipoprotein receptor 0.0593 1.22Microtubule-associated protein 2 0.0003 0.79 Microtubule-associatedprotein tau 0.0021 0.86 MAP kinase-interacting serine/threonine kinase 10.0459 0.88 NCK-associated protein 1 0.0000 0.75 Polyhomeotic-like 2(Drosophila) 0.0055 0.87 Phosphatidic acid phosphatase type 2B 0.02320.82 Protein phosphatase 1A, magnesium-dependent, alpha isoform 0.14250.93 Protein phosphatase 2, regulatory subunit B (B56), beta isoform0.0059 0.87 Protein phosphatase 4, regulatory subunit 1 0.0147 0.86Protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue 0.00260.91 specific extinguisher 1) Pleckstrin and Sec7 domain protein 0.07830.88 Pleiotrophin 0.0093 0.77 Protein tyrosine phosphatase, receptortype, M 0.0009 0.84 RAB22A, member RAS oncogene family 0.7950 0.98Solute carrier family 1 (glial high affinity glutamate transporter),0.0091 0.76 member 3 Sintaxin 10 0.0028 0.84 Synapsin II 0.1656 0.92Tachykinin, precursor 1 0.0297 0.83 Tachykinin, precursor 1 0.0617 0.82Adrenergic alpha1A receptor 0.0206 0.84 Adrenergic alpha1B receptor0.0002 0.73 Adrenergic alpha2A receptor 0.5100 0.93 Brain-derivedneurotrophic factor 0.2077 0.83 Corticotropin releasing hormone 0.37530.87 Corticotropin releasing hormone receptor 1 0.1483 0.83Corticotropin releasing hormone receptor 2 0.7045 1.04 Serotoninreceptor 1A 0.0005 0.79 Serotonin receptor 1B 0.1175 0.92 Serotoninreceptor 1E 0.8936 1.01 Serotonin receptor 2A 0.2935 0.92 Serotoninreceptor 2C 0.6676 1.10 Tyrosine kinase receptor type 1 0.5002 0.89Tyrosine kinase receptor type 3 0.0864 0.90 Opioid receptor, mu 1 0.93091.01

TABLE 4 Neuron-specific promoters: sequence information NCBI Buildnumber is as shown in the Table. NCBI Genomic Refseq Promoter GenomicPromoter Genomic Gene (Including introns Refseq (5′ Refseq (5′ ID SymbolDescription RNA Refseq Protein Refseq and exons) up-stream 10 kb)up-stream 100 kb) mouse DBH dopamine beta NM_138942.3 NP_620392.2NC_000068.7 (This gene is on + (This gene is on + 13166 hydroxylase(27165508- strand) (27155508- strand) (27065508- 27183204) 27165508)27165508) (NCBI build 38.1) human DBH dopamine beta- NM_000787.3NP_000778.3 NC_000009.11 (This gene is on + (This gene is on + 1621hydroxylase (136501485- strand) strand) (136401485- (dopamine beta-136524466) (136491485136501485) 136501485) monooxygenase) NCBI build37.3 mouse Eno2 enolase 2, gamma NM_013509.2 NP_038537.1 NC_000072.6(This gene is on − (This gene is on − 13807 neuronal (124760055- strand)(124779509- strand) (124869509- 124769509) 124769509) 124769509) NCBIbuild 38.1 human ENO2 enolase 2 (gamma, NM_001975.2 NP_001966.1NC_000012.11 (This gene is on + (This gene is on + 2026 neuronal)(7023614- strand) (7013614- strand) (6923614- 7032859) 7023614) 7023614)NCBI Build 37.3 mouse Lmo1 LIM domain only 1 NM_057173.2 NP_476514.1NC_000073.6 (This gene is on − (This gene is on − 109594 (109138572-strand) (109170308- strand) (109170308- 109170308) 109180308) 109270308)NCBI build 38.1 human LMO1 LIM domain only 1 NM_002315.1 NP_002306.1NC_000011.9 (This gene is on − (This gene is on − 4004 (rhombotin 1)(8245857- strand) (8285406- strand) (8285406- 8285406) 8295405) 8385405)NCBI Build 37.3 mouse Nefl neurofilament, light NM_010910.1 NP_035040.1NC_000080.6 (This gene is on + (This gene is on + 18039 polypeptide(68083884- strand) (68083884- strand) (68083884- 68087745) 68073884)67983884) NCBI 38.1 human NEFL neurofilament, light NM_006158.3NP_006149.2 NC_000008.10 (This gene is on − (This gene is on − 4747polypeptide (24808468- strand) (24814131- strand) (24814131- 24814131)24824130) 24914130) NCBI 37.3 mouse Pgk1 phosphoglycerate NM_008828.2NP_032854.2 NC_000086.7 (This gene is on + (This gene is on + 18655kinase 1 (106187124- strand) (106187124- strand) (106187124- 106203699)106177124) 106087124) NCBI 38.1 human PGK1 phosphoglycerate NM_000291.3NP_000282.1 NC_000023.10 (This gene is on + (This gene is on + 5230kinase 1 (77359665- strand) (77349665- strand) (77259665- 77382323)77359664) 77359664) NCBI 37.2 mouse Syn1 synapsin I NM_013680.4NP_038708.3 NC_000086.7 (This gene is on − (This gene is on − 20964(20860511- strand) (20930918- strand) (201020918- 20920918) 20920918)20920918) NCBI build 38.1 mouse Syn1 synapsin I NM_001110780.1NP_001104250.1 NC_000086.7 (This gene is on − (This gene is on − 20964(20860511- strand) (20930918- strand) (201020918- 20920918) 20920918)20920918) NCBI build 38.1 human SYN1 synapsin I NM_133499.2 NP_598006.1NC_000023.10 (This gene is on − (This gene is on − 6853 (47431299-strand) (47479256- strand) (47479256- 47479255) 47489255) 47579255) NCBIbuild 37.3 human SYN1 synapsin 1 NM_006950.3 NP_008881.2 NC_000023.10(This gene is on − (This gene is on − 6853 (47431299- strand) (47479256-strand) (47479256- 47479255) 47489255) 47579255) NCBI build 37.3 mouseThy1 thymus cell antigen NM_009382.3 NP_033408.1 NC_000075.6 (This geneis on + (This gene is on + 21838 1, theta (44043384- strand) (44033384-strand) (43943384- 44048579) 44043384) 44043384) NCBI Build 38.1 humanTHY1 Thy-1 cell surface NM_006288.3 NP_006279.2 NC_000011.9 (This geneis on − (This gene is on − 7070 antigen (119288654- strand) (119294246-strand) (119294246- 119294245) 119304245) 119394245) NCBI Build 37.3

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety for all purposes,particularly for the disclosure referenced herein.

REFERENCE LIST

-   Belmaker R H, Agam G (2008) Major depressive disorder. N Engl J Med    358:55-68.-   Calvet L, Geoerger B, Regairaz M, Opolon P, Machet L, Morizet J,    Joseph J M, Elie N, Vassal G (2006) Pleiotrophin, a candidate gene    for poor tumor vasculature and in vivo neuroblastoma sensitivity to    irinotecan. Oncogene 25:3150-3159.-   Chen H, Gordon M S, Campbell R A, Li M, Wang C S, Lee H J, Sanchez    E, Manyak S J, Gui D, Shalitin D, Said J, Chang Y, Deuel T F,    Baritaki S, Bonavida B, Berenson J R (2007) Pleiotrophin is highly    expressed by myeloma cells and promotes myeloma tumor growth. Blood    110:287-295.-   Christensen J G, Zou H Y, Arango M E, Li Q, Lee J H, McDonnell S R,    Yamazaki S, Alton G R, Mroczkowski B, Los G (2007) Cytoreductive    antitumor activity of PF-2341066, a novel inhibitor of anaplastic    lymphoma kinase and c-Met, in experimental models of anaplastic    large-cell lymphoma. Mol Cancer Ther 6:3314-3322.-   DellaGioia N, Hannestad J (2010) A critical review of human    endotoxin administration as an experimental paradigm of depression.    Neurosci Biobehav Rev 34:130-143.-   Deuel T F, Zhang N, Yeh H J, Silos-Santiago I, Wang Z Y (2002)    Pleiotrophin: a cytokine with diverse functions and a novel    signaling pathway. Arch Biochem Biophys 397:162-171.-   Diamantopoulou Z, Bermek O, Polykratis A, Hamma-Kourbali Y, Delbe J,    Courty J, Katsoris P (2010) A Pleiotrophin C-terminus peptide    induces anti-cancer effects through RPTPbeta/zeta. Mol Cancer 9:224.-   Dunn A J, Swiergiel A H, de BR (2005) Cytokines as mediators of    depression: what can we learn from animal studies? Neurosci Biobehav    Rev 29:891-909.-   Furuta M, Shiraishi T, Okamoto H, Mineta T, Tabuchi K, Shiwa    M (2004) Identification of pleiotrophin in conditioned medium    secreted from neural stem cells by SELDI-TOF and SELDI-tandem mass    spectrometry. Brain Res Dev Brain Res 152:189-197.-   Hamma-Kourbali Y, Bernard-Pierrot I, Heroult M, Dalle S, Caruelle D,    Milhiet P E, Fernig D G, Delbe J, Courty J (2008) Inhibition of the    mitogenic, angiogenic and tumorigenic activities of pleiotrophin by    a synthetic peptide corresponding to its C-thrombospondin repeat-I    domain. J Cell Physiol 214:250-259.-   Heinrich M C, Corless C L, Demetri G D, Blanke C D, von M M, Joensuu    H, McGreevey L S, Chen C J, Van den Abbeele A D, Druker B J, Kiese    B, Eisenberg B, Roberts P J, Singer S, Fletcher C D, Silberman S,    Dimitrijevic S, Fletcher J A (2003) Kinase mutations and imatinib    response in patients with metastatic gastrointestinal stromal tumor.    J Clin Oncol 21:4342-4349.-   Herry C, Garcia R (2002) Prefrontal cortex long-term potentiation,    but not long-term depression, is associated with the maintenance of    extinction of learned fear in mice. J Neurosci 22:577-583.-   Holderbach R, Clark K, Moreau J L, Bischofberger J, Normann C (2007)    Enhanced long-term synaptic depression in an animal model of    depression. Biol Psychiatry 62:92-100.-   Huang P, Ramphal J, Wei J, Liang C, Jallal B, McMahon G, Tang    C (2003) Structure-based design and discovery of novel inhibitors of    protein tyrosine phosphatases. Bioorg Med Chem 11:1835-1849.-   Kadomatsu K, Muramatsu T (2004) Midkine and pleiotrophin in neural    development and cancer. Cancer Lett 204:127-143.-   Krishnan V, Nestler E J (2008) The molecular neurobiology of    depression. Nature 455:894-902.-   Krishnan V, Nestler E J (2010) Linking molecules to mood: new    insight into the biology of depression. Am J Psychiatry    167:1305-1320.-   Lee S, Jeong J, Kwak Y, Park S K (2010) Depression research: where    are we now? Mol Brain 3:8.-   Lorente G, Nelson A, Mueller S, Kuo J, Urfer R, Nikolich K, Foehr E    D (2005) Functional comparison of long and short splice forms of    RPTPbeta: implications for glioblastoma treatment. Neuro Oncol    7:154-163.-   Mikelis C, Lamprou M, Koutsioumpa M, Koutsioubas A G, Spyranti Z,    Zompra A A, Spiliopoulos N, Vradis A A, Katsoris P, Spyroulias G A,    Cordopatis P, Courty J, Papadimitriou E (2011) A peptide    corresponding to the C-terminal region of pleiotrophin inhibits    angiogenesis in vivo and in vitro. J Cell Biochem 112:1532-1543.-   Milner P G, Shah D, Veile R, Donis-Keller H, Kumar B V (1992)    Cloning, nucleotide sequence, and chromosome localization of the    human pleiotrophin gene. Biochemistry 31:12023-12028.-   Pavlov I, Voikar V, Kaksonen M, Lauri S E, Hienola A, Taira T,    Rauvala H (2002) Role of heparin-binding growth-associated molecule    (HB-GAM) in hippocampal LTP and spatial learning revealed by studies    on overexpressing and knockout mice. Mol Cell Neurosci 20:330-342.

Sabbatini P, Korenchuk S, Rowand J L, Groy A, Liu Q, Leperi D, Atkins C,Dumble M, Yang J, Anderson K, Kruger R G, Gontarek R R, Maksimchuk K R,Suravajjala S, Lapierre R R, Shotwell J B, Wilson J W, Chamberlain S D,Rabindran S K, Kumar R (2009) GSK1838705A inhibits the insulin-likegrowth factor-1 receptor and anaplastic lymphoma kinase and showsantitumor activity in experimental models of human cancers. Mol CancerTher 8:2811-2820.

-   Stewart C A, Reid I C (2002) Antidepressant mechanisms: functional    and molecular correlates of excitatory amino acid neurotransmission.    Mol Psychiatry 7 Suppl 1:S15-S22.-   Sullivan P F, Neale M C, Kendler K S (2000) Genetic epidemiology of    major depression: review and meta-analysis. Am J Psychiatry    157:1552-1562.-   Tesseur I, Van D J, Spittaels K, Van Den Haute C, Moechars D, Van L    F (2000) Expression of human apolipoprotein E4 in neurons causes    hyperphosphorylation of protein tau in the brains of transgenic    mice. Am J Pathol 156:951-964.-   Vandesompele J, De P K, Pattyn F, Poppe B, Van R N, De P A, Speleman    F (2002) Accurate normalization of real-time quantitative RT-PCR    data by geometric averaging of multiple internal control genes.    Genome Biol 3:RESEARCH0034.-   Wong M L, Licinio J (2004) From monoamines to genomic targets: a    paradigm shift for drug discovery in depression. Nat Rev Drug Discov    3:136-151.-   Yao J, Zhang M, Ma Q Y, Wang Z, Wang L C, Zhang D (2011)    PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and    inhibits neurite outgrowth of DRG. World J Gastroenterol    17:2667-2673.

1. A method of assessing the presence of or susceptibility to anxietyand/or depression, in a human test subject, comprising: detecting and/ordetermining the amount of a Pleiotrophin (PTN) polypeptide in a samplewhich has been obtained from said test subject; and comparing saidamount of the PTN polypeptide with one or more reference valuescorresponding to the amount of the PTN polypeptide in a control sampleobtained from a control subject not having a neuropsychiatric illness,wherein an increase of at least 10% in said amount of the PTNpolypeptide in the sample which has been obtained from said test subjectcompared to said reference value indicates the presence of orsusceptibility to anxiety and/or depression in the subject, and whereinsaid sample is selected from the group consisting of: blood, plasma,serum, cerebrospinal fluid (CSF) and central nervous system tissue. 2.The method of claim 1, wherein the test subject has not previously beendiagnosed as having a neuropsychiatric illness.
 3. The method of claim1, wherein the test subject has previously been diagnosed as having aneuropsychiatric illness, and wherein said method is a method ofassessing the stage and/or severity of the illness, to select thepatients that will benefit from treatment or to monitor the effect of atreatment administered to the test subject.
 4. The method of claim 1,wherein said depression is major depression.
 5. The method of claim 1,wherein the amount of PTN polypeptide in the sample which has beenobtained from said test subject is determined to be at least 20% higherthan said reference value.
 6. The method of claim 1, wherein the methodcomprises contacting the sample with one or more antibodies specific forPTN polypeptide, allowing said one or more antibodies to form a complexwith the PTN polypeptide, and quantifying the antibody-PIN proteincomplex, thereby determining the amount of PTN polypeptide in thesample.
 7. The method of claim 6, wherein the method employs a techniqueselected from the group consisting of: Western blot, Western transfer,Enzyme-Linked Immunosorbent Assay (ELISA), Radioimmunoassay (RIA),competitive enzyme immunoassay (EIA) and double antibody sandwich-ELISA(DAS-ELISA).
 8. The method of claim 1, wherein the method comprisesextracting protein from the sample and mixing the protein extract withone or more antibodies specific for PTN polypeptide, allowing said oneor more antibodies to form a complex with the PTN polypeptide andquantifying the antibody-PTN protein complex, thereby determining theamount of PTN polypeptide in the sample.
 9. The method of claim 6,wherein at least one of said one or more antibodies specific for PTNpolypeptide is a goat polyclonal antibody available as product AF-252-PBfrom R&D Systems.
 10. The method of claim 1, wherein determining theamount of PTN polypeptide in the sample comprises subjecting the sample,or a protein extract obtained from the sample, to mass spectrometryanalysis and quantifying the amount of PTN polypeptide detected.
 11. Amethod of assessing the presence of or susceptibility to anxiety and/ordepression, in a human test subject, comprising: detecting and/ordetermining the amount of an mRNA or cDNA encoding a Pleiotrophin (PTN)polypeptide in a sample which has been obtained from said test subject;and comparing said amount of the mRNA or cDNA encoding the PTNpolypeptide with one or more reference values corresponding to theamount of the mRNA or cDNA encoding the PTN polypeptide in a controlsample obtained from a control subject not having a neuropsychiatricillness, wherein an increase of at least 10% in said amount of the mRNAor cDNA encoding the PTN polypeptide in the sample which has beenobtained from said test subject compared to said reference valueindicates the presence of or susceptibility to anxiety and/or depressionin the subject, and wherein said sample is selected from the groupconsisting of: blood, plasma, serum, cerebrospinal fluid (CSF) andcentral nervous system tissue.
 12. The method of claim 11, wherein thetest subject has not previously been diagnosed as having aneuropsychiatric illness.
 13. The method of claim 11, wherein the testsubject has previously been diagnosed as having a neuropsychiatricillness, and wherein said method is a method of assessing the stageand/or severity of the illness, to select the patients that will benefitfrom treatment or to monitor the effect of a treatment administered tothe test subject.
 14. The method of claim 11, wherein said depression ismajor depression.
 15. The method of claim 11, wherein the amount of themRNA or cDNA encoding the PTN polypeptide in the sample which has beenobtained from said test subject is determined to be at least 20% higherthan said reference value.
 16. The method of claim 11, wherein themethod comprises extracting RNA from the sample, reverse transcribingthe RNA to form cDNA, and amplifying the PTN polypeptide-encoding cDNAby polymerase chain reaction (PCR) using oligonucleotide primers,thereby determining the amount of the mRNA or cDNA encoding the PTNpolypeptide in the sample.
 17. The method of claim 16, wherein said PCRis real-time quantitative PCR.